Amidino compounds useful as nitric oxide synthase inhibitors

ABSTRACT

The present invention relates to amidino compounds and salts and prodrugs thereof. In another embodiment the present invention also provides a use of the present compounds in therapy, particular as nitric oxide synthase inhibitors. In a further embodiment, the present invention provides methods of making the amidino compounds.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a division of U.S. Ser. No. 10/321,969, filed Dec.17, 2002, now abandoned which is a division of U.S. Ser. No. 09/816,575,filed Mar. 23, 2001, U.S. Pat. No. 6,586,474, which claims the benefitof priority of U.S. Provisional Patent Application No. 60/191,923 filedMar. 24, 2000, herein incorporated by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to amidino compounds and their use intherapy, in particular their use as nitric oxide synthase inhibitors.

2. Related Art

It has been known since the early 1980's that the vascular relaxationcaused by acetylcholine is dependent on the vascular endothelium. Theendothelium-derived relaxing factor (EDRF), now known to be nitric oxide(NO) is generated in the vascular endothelium by nitric oxide synthase(NOS). The activity of NO as a vasodilator has been known for well over100 years. In addition, NO is the active species deriving fromamylnitrite, glyceryltrinitrate and other nitrovasodilators. Theidentification of EDRF as NO has coincided with the discovery of abiochemical pathway by which NO is synthesized from the amino acidL-arginine by the enzyme NO synthase.

Nitric oxide is an endogenous stimulator of the soluble guanylatecyclase. In addition to endothelium-dependent relaxation, NO is involvedin a number of biological actions including cytotoxicity of phagocyticcells and cell-to-cell communication in the central nervous system.

There are at least three types of NO synthase as follows:

(i) a constitutive, Ca⁺⁺/calmodulin dependent enzyme, located in theendothelium, that releases NO in response to receptor or physicalstimulation.

(ii) a constitutive, Ca⁺⁺/calmodulin dependent enzyme, located in thebrain, that releases NO in response to receptor or physical stimulation.

(iii) a Ca⁺⁺ independent enzyme which is induced after activation ofvascular smooth muscle, macrophages, endothelial cells, and a number ofother cells by endotoxin and cytokines. Once expressed this induciblenitric oxide synthase (hereinafter “iNOS”) generates NO continuously forlong periods.

The NO released by each of the two constitutive enzymes acts as atransduction mechanism underlying several physiological responses. TheNO produced by the inducible enzyme is a cytotoxic molecule for tumorcells and invading microorganisms. It also appears that adverse effectsof excess NO production, in particular pathological vasodilation andtissue damage, may result largely from the NO synthesized by iNOS.

There is a growing body of evidence that NO may be involved in thedegeneration of cartilage which takes place as a result of certainconditions such as arthritis and it is also known that NO synthesis isincreased in rheumatoid arthritis and in osteoarthritis.

Some of the NO synthase inhibitors proposed for therapeutic use arenon-selective; they inhibit both the constitutive and the inducible NOsynthases. Use of such a non-selective NO synthase inhibitor requiresthat great care be taken in order to avoid the potentially seriousconsequences of over-inhibition of the constitutive NO-synthaseincluding hypertension and possible thrombosis and tissue damage. Inparticular, in the case of the therapeutic use of L-NMMA for thetreatment of toxic shock it has been recommended that the patient mustbe subject to continuous blood pressure monitoring throughout thetreatment. Thus, while non-selective NO synthase inhibitors havetherapeutic utility provided that appropriate precautions are taken, NOsynthase inhibitors which are selective in the sense that they inhibitthe inducible NO synthase to a considerably greater extent than theconstitutive isoforms of NO synthase would be of even greatertherapeutic benefit and easier to use (S. Moncada and E. Higgs, FASEBJ., 9, 1319-1330, 1995).

The following individual publications disclose compounds that inhibitnitric oxide synthesis and preferentially inhibit the inducible isoformof nitric oxide synthase:

-   PCT Patent Application No. WO 96/35677.-   PCT Patent Application No. WO 96/33175.-   PCT Patent Application No. WO 96/15120.-   PCT Patent Application No. WO 95/11014.-   PCT Patent Application No. WO 95/11231.-   PCT Patent Application No. WO 99/46240.-   PCT Patent Application No. WO 95/24382.-   PCT Patent Application No. WO 94/12165.-   PCT Patent Application No. WO 94/14780.-   PCT Patent Application No. WO 93/13055.-   PCT Patent Application No. WO 99/62875.-   European Patent No. EP0446699A1.-   U.S. Pat. No. 5,132,453.-   U.S. Pat. No. 5,684,008.-   U.S. Pat. No. 5,830,917.-   U.S. Pat. No. 5,854,251.-   U.S. Pat. No. 5,863,931.-   U.S. Pat. No. 5,919,787.-   U.S. Pat. No. 5,945,408.-   U.S. Pat. No. 5,981,511.

PCT Patent Application No. WO 95/25717 discloses certain amidinoderivatives as being useful in inhibiting inducible nitric oxidesynthase.

PCT Patent Application No. WO 99/62875 discloses further amidinocompounds as being useful in inhibiting inducible nitric oxide synthase.

SUMMARY OF THE INVENTION

Compounds have now been found which have the advantage of being veryefficacious as iNOS inhibitors in the human cartilage explant assay, amodel for osteoarthritis. At the same time the compounds of the presentinvention are surprisingly unable to penetrate certain non-target organsin test systems, especially in comparison to the compounds of WO95/25717. This surprising differentiation in expected access between thetarget organ (cartilage) and other organs is an unexpected advantage forthe compounds of the present invention.

In a broad aspect, the present invention is directed to novel compounds,pharmaceutical compositions and methods of using said compounds andcompositions for inhibiting or modulating nitric oxide synthesis in asubject in need of such inhibition or modulation by administering acompound which preferentially inhibits or modulates the inducibleisoform of nitric oxide synthase over the constitutive isoforms ofnitric oxide synthase. It is also another object of the presentinvention to lower nitric oxide levels in a subject in need of suchlowering. The present compounds possess useful nitric oxide synthaseinhibiting activity, and are expected to be useful in the treatment orprophylaxis of a disease or condition in which the synthesis orover-synthesis of nitric oxide forms a contributory part.

In one embodiment, the present invention provides a compound or a saltthereof, the compound having a structure corresponding to Formula 1:

wherein:

-   -   X is selected from the group consisting of —S—, —S(O)—, and        —S(O)₂—;    -   R² is selected from the group consisting of C₁-C₆ alkyl, C₂-C₆        alkenyl, C₂-C₆ alkynyl, C₁-C₅ alkoxy-C₁ alkyl, and C₁-C₅        alkylthio-C₁ alkyl;    -   with respect to R³ and R⁸:    -   R⁸ is selected from the group consisting of —OR¹⁴ and        —N(R¹⁵)(R¹⁶); and R³ is selected from the group consisting of        —H, —OH, —C(O)—R¹⁷, —C(O)—O—R¹⁸, and —C(O)—S—R¹⁹; or    -   R⁸ is —N(R²⁰)—, and R³ is —C(O)—, wherein R⁸ and R³ together        with the atoms to which they are attached form a ring; or    -   R⁸ is —O—, and R³ is —C(R²¹)(R²²)—, wherein R⁸ and R³ together        with the atoms to which they are attached form a ring;    -   if R³ is —C(R²¹)(R²²)—, then R²³ is —C(O)—O—R²³; otherwise R⁴ is        —H;    -   R¹, R⁵, R⁶, and R⁷ independently are selected from the group        consisting of —H, halogen, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆        alkynyl, and C₁-C₅ alkoxy-C₁ alkyl;    -   R⁹ and R¹⁰ independently are selected from the group consisting        of —H, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, and C₁-C₅        alkoxy-C₁ alkyl;    -   with respect to R¹¹ and R¹²:    -   R¹¹ is selected from the group consisting of —H, —OH,        —C(O)—O—R²⁴, and —C(O)—S—R²⁵; and R¹² is selected from the group        consisting of —H, —OH, —C(O)—O—R²⁶, and —C(O)—S—R²⁷; or    -   R¹¹ is —O—, and R¹² is —C(O)—, wherein R¹¹ and R¹² together with        the atoms to which they are attached form a ring; or    -   R¹¹ is —C(O)—, and R¹² is —O—, wherein R¹¹ and R¹² together with        the atoms to which they are attached form a ring; and    -   R¹³ is C₁ alkyl;    -   R¹⁴ is selected from the group consisting of —H and C₁-C₆ alkyl;        wherein when R¹⁴ is C₁-C₆ alkyl, R¹⁴ is optionally substituted        by one or more moieties selected from the group consisting of        cycloalkyl, heterocyclyl, aryl, and heteroaryl;    -   with respect to R¹⁵ and R¹⁶:    -   R¹⁵ is selected from the group consisting of —H, alkyl, and        alkoxy; and R¹⁶ is selected from the group consisting of —H,        —OH, alkyl, alkoxy, —C(O)—R^(27a), —C(O)—O—R²⁸, and —C(O)—S—R²⁹;        wherein when R¹⁵ and R¹⁶ independently are alkyl or alkoxy, R¹⁵        and R¹⁶ independently are optionally substituted with one or        more moieties selected from the group consisting of cycloalkyl,        heterocyclyl, aryl, and heteroaryl; or    -   R¹⁵ is —H; and R¹⁶ is selected from the group consisting of        cycloalkyl, heterocyclyl, aryl, and heteroaryl;    -   R¹⁷, R¹⁸, R¹⁹, R²⁰, R²¹, R²², R²³, R²⁴, R²⁵, R²⁶, R²⁷, R^(27a),        R²⁸, and R²⁹ independently are selected from the group        consisting of —H and alkyl, which is optionally substituted by        one or more moieties selected from the group consisting of        cycloalkyl, heterocyclyl, aryl, and heteroaryl; and    -   when any of R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹²,        R¹³, R¹⁴, R¹⁵, R¹⁶, R¹⁷, R¹⁸, R¹⁹, R²⁰, R²¹, R²², R²³, R²⁴, R²⁵,        R²⁶, R²⁷, R^(27a), R²⁸, and R²⁹ independently is a moiety        selected from the group consisting of alkyl, alkenyl, alkynyl,        alkoxy, alkylthio, cycloalkyl, heterocyclyl, aryl, and        heteroaryl, then the moiety is optionally substituted by one or        more substituent selected from the group consisting of —OH,        alkoxy, and halogen.

Another embodiment provides a method for the treatment or prevention ofan inflammation-related disorder wherein the method comprises treating asubject in need thereof with an inflammation-related disorder treatingor preventing amount of a compound of the present invention.

In yet another embodiment the present invention provides a method forthe preparation of a compound of Formula 1, wherein the method comprisestreating a diamine compound having a structure corresponding to Formula22:

or a salt thereof, with an alkyl acetimidate having a structurecorresponding to Formula 23:

wherein R³¹ is C₁-C₆ alkyl. The treating can, if desired, be performedin the presence of an acid or a base, preferably in the presence of abase.

In a further embodiment of the present invention provides a method forthe preparation of a diamine compound having a structure correspondingto Formula 22:

or a salt thereof, wherein R³⁰ is selected from the group consisting of—H, —OH, —C(O)—R¹⁷, —C(O)—O—R¹⁸, and —C(O)—S—R¹⁹, and the othersubstituents are as defined above, wherein the method comprises treatinga protected diamine compound having the structure corresponding toFormula 24:

or a salt thereof, wherein R³³ is selected from the group consisting of—H and a protected amino group; and R³² is a protected amino group; andR¹⁴ is selected from the group consisting of —H and C₁-C₆ alkyl; whereinwhen R¹⁴ is C₁-C₆ alkyl, R¹⁴ is optionally substituted by one or moremoieties selected from the group consisting of cycloalkyl, heterocyclyl,aryl, and heteroaryl; wherein the treating is performed with adeprotecting reagent, thereby producing the diamine compound.

DETAILED DESCRIPTION OF THE INVENTION

Compounds of Formula 1 will be useful for treating, among other things,inflammation in a subject, or for treating other nitric oxidesynthase-mediated disorders, such as, as an analgesic in the treatmentof pain and headaches, or as an antipyretic for the treatment of fever.For example, compounds of the present invention will be useful to treatarthritis, including but not limited to rheumatoid arthritis,spondyloarthropathies, gouty arthritis, osteoarthritis, systemic lupuserythematosus, juvenile arthritis, acute rheumatic arthritis,enteropathic arthritis, neuropathic arthritis, psoriatic arthritis, andpyogenic arthritis. Conditions in which the compounds of the presentinvention will provide an advantage in inhibiting NO production fromL-arginine include arthritic conditions.

Compounds of the invention will be further useful in the treatment ofasthma, bronchitis, menstrual cramps (e.g., dysmenorrhea), prematurelabor, tendinitis, bursitis, skin-related conditions such as psoriasis,eczema, burns, sunburn, dermatitis, pancreatitis, hepatitis, and frompost-operative inflammation including from ophthalmic surgery such ascataract surgery and refractive surgery. Compounds of the invention alsowould be useful to treat gastrointestinal conditions such asinflammatory bowel disease, Crohn's disease, gastritis, irritable bowelsyndrome and ulcerative colitis. Compounds of the invention would beuseful for the prevention or treatment of cancer, such as colorectalcancer, and cancer of the breast, lung, prostate, bladder, cervix andskin. Compounds of the invention would be useful in treatinginflammation and tissue damage in such diseases as vascular diseases,migraine headaches, periarteritis nodosa, thyroiditis, aplastic anemia,Hodgkin's disease, sclerodoma, rheumatic fever, type I diabetes,neuromuscular junction disease including myasthenia gravis, white matterdisease including multiple sclerosis, sarcoidosis, nephrotic syndrome,Behcet's syndrome, polymyositis, gingivitis, nephritis,hypersensitivity, swelling occurring after injury, myocardial ischemia,and the like. The compounds would also be useful in the treatment ofophthalmic diseases, such as glaucoma, retinitis, retinopathies,uveitis, ocular photophobia, and of inflammation and pain associatedwith acute injury to the eye tissue. Of particular interest among theuses of the present inventive compounds is the treatment of glaucoma,especially where symptoms of glaucoma are caused by the production ofnitric oxide, such as in nitric oxide-mediated nerve damage. Thecompounds would also be useful in the treatment of pulmonaryinflammation, such as that associated with viral infections and cysticfibrosis. The compounds would also be useful for the treatment ofcertain central nervous system disorders, such as cortical dementiasincluding Alzheimer's disease, and central nervous system damageresulting from stroke, ischemia and trauma. The compounds of theinvention are useful as anti-inflammatory agents, such as for thetreatment of arthritis, with the additional benefit of havingsignificantly less harmful side effects. These compounds would also beuseful in the treatment of allergic rhinitis, respiratory distresssyndrome, endotoxin shock syndrome, and atherosclerosis. The compoundswould also be useful in the treatment of pain, but not limited topostoperative pain, dental pain, muscular pain, and pain resulting fromcancer. The compounds would be useful for the prevention of dementias,such as Alzheimer's disease.

Besides being useful for human treatment, these compounds are alsouseful for veterinary treatment of companion animals, exotic animals andfarm animals, including mammals, rodents, and the like. More preferredanimals include horses, dogs, and cats.

The present compounds may also be used in co-therapies, partially orcompletely, in place of other conventional antiinflammatory therapies,such as together with steroids, NSAIDs, COX-2 selective inhibitors,5-lipoxygenase inhibitors, LTB₄ antagonists and LTA4 hydrolaseinhibitors.

Other conditions in which the compounds of the present invention willprovide an advantage in inhibiting NO inhibition include cardiovascularischemia, diabetes (type I or type II), congestive heart failure,myocarditis, atherosclerosis, migraine, glaucoma, aortic aneurysm,reflux esophagitis, diarrhea, irritable bowel syndrome, cystic fibrosis,emphysema, asthma, bronchiectasis, hyperalgesia (allodynia), cerebralischemia (both focal ischemia, thrombotic stroke and global ischemia(for example, secondary to cardiac arrest), multiple sclerosis and othercentral nervous system disorders mediated by NO, for example Parkinson'sdisease. Further neurodegenerative disorders in which NO inhibition maybe useful include nerve degeneration or nerve necrosis in disorders suchas hypoxia, hypoglycemia, epilepsy, and in cases of central nervoussystem (CNS) trauma (such as spinal cord and head injury), hyperbaricoxygen convulsions and toxicity, dementia e.g. pre-senile dementia, andAIDS-related dementia, cachexia, Sydenham's chorea, Huntington'sdisease, Amyotrophic Lateral Sclerosis, Korsakoff's disease, imbecilityrelating to a cerebral vessel disorder, sleeping disorders,schizophrenia, depression, depression or other symptoms associated withPremenstrual Syndrome (PMS), anxiety and septic shock.

The compounds of the present invention will also be useful in thetreatment of pain including somatogenic (either nociceptive orneuropathic), both acute and chronic. A nitric oxide inhibitor could beused in any situation including neuropathic pain that a common NSAID oropioid analgesic would traditionally be administered.

Still other disorders or conditions which will be advantageously treatedby the compounds of the present invention include treatment ofprevention of opiate tolerance in patients needing protracted opiateanalgesics, and benzodiazepine tolerance in patients takingbenzodiazepines, and other addictive behavior, for example, nicotineaddiction, alcoholism, and eating disorders. The compounds and methodsof the present invention will also be useful in the treatment orprevention of drug withdrawal symptoms, for example treatment orprevention of symptoms of withdrawal from opiate, alcohol, or tobaccoaddiction. The present inventive compounds may also be useful to preventtissue damage when therapeutically combined with antibacterial orantiviral agents.

The compounds of the present invention will also be useful in inhibitingNO production from L-arginine including systemic hypotension associatedwith septic and/or toxic hemorrhagic shock induced by a wide variety ofagents; therapy with cytokines such as TNF, IL-1 and IL-2; and as anadjuvant to short term immunosuppression in transplant therapy.

The present invention is further directed to the use of the compounds ofthe present invention for the treatment and prevention of neoplasias.The neoplasias that will be treatable or preventable by the compoundsand methods of the present invention include brain cancer, bone cancer,a leukemia, a lymphoma, epithelial cell-derived neoplasia (epithelialcarcinoma) such as basal cell carcinoma, adenocarcinoma,gastrointestinal cancer such as lip cancer, mouth cancer, esophogealcancer, small bowel cancer and stomach cancer, colon cancer, livercancer, bladder cancer, pancreas cancer, ovary cancer, cervical cancer,lung cancer, breast cancer and skin cancer, such as squamous cell andbasal cell cancers, prostate cancer, renal cell carcinoma, and otherknown cancers that effect epithelial cells throughout the body.Preferably, the neoplasia is selected from gastrointestinal cancer,liver cancer, bladder cancer, pancreas cancer, ovary cancer, prostatecancer, cervical cancer, lung cancer, breast cancer and skin cancer,such as squamous cell and basal cell cancers. The present compounds andmethods can also be used to treat the fibrosis which occurs withradiation therapy. The present compounds and methods can be used totreat subjects having adenomatous polyps, including those with familialadenomatous polyposis (FAP). Additionally, the present compounds andmethods can be used to prevent polyps from forming in patients at riskof FAP.

Conjunctive treatment of a compound of the present invention withanother antineoplastic agent will produce a synergistic effect oralternatively reduce the toxic side effects associated with chemotherapyby reducing the therapeutic dose of the side effect-causing agent neededfor therapeutic efficacy or by directly reducing symptoms of toxic sideeffects caused by the side effect-causing agent. A compound of thepresent invention will further be useful as an adjunct to radiationtherapy to reduce side effects or enhance efficacy. In the presentinvention, another agent which can be combined therapeutically with acompound of the present invention includes any therapeutic agent whichis capable of inhibiting the enzyme cyclooxygenase-2 (“COX-2”).Preferably such COX-2 inhibiting agents inhibit COX-2 selectivelyrelative to the enzyme cyclooxygenase-1 (“COX-1”). Such a COX-2inhibitor is known as a “COX-2 selective inhibitor”. More preferably, acompound of the present invention can be therapeutically combined with aCOX-2 selective inhibitor wherein the COX-2 selective inhibitorselectively inhibits COX-2 at a ratio of at least 10:1 relative toinhibition of COX-1, more preferably at least 30:1, and still morepreferably at least 50:1 in an in vitro test. COX-2 selective inhibitorsuseful in therapeutic combination with the compounds of the presentinvention include celecoxib, valdecoxib, deracoxib, etoricoxib,rofecoxib, ABT-963(2-(3,4-difluorophenyl)-4-(3-hydroxy-3-methyl-1-butoxy)-5-[4-(methylsulfonyl)phenyl-3(2H)-pyridazinone;described in PCT Patent Application No. WO 00/24719), or meloxicam. Acompound of the present invention can also be advantageously used intherapeutic combination with a prodrug of a COX-2 selective inhibitor,for example parecoxib.

Another chemotherapeutic agent which will be useful in combination witha compound of the present invention can be selected, for example, fromthe following non-comprehensive and non-limiting list:

Alpha-difluoromethylornithine (DFMO), 5-FU-fibrinogen, acanthifolicacid, aminothiadiazole, brequinar sodium, carmofur, Ciba-GeigyCGP-30694, cyclopentyl cytosine, cytarabine phosphate stearate,cytarabine conjugates, Lilly DATHF, Merrel Dow DDFC, dezaguanine,dideoxycytidine, dideoxyguanosine, didox, Yoshitomi DMDC, doxifluridine,Wellcome EHNA, Merck & Co. EX-015, fazarabine, floxuridine, fludarabinephosphate, 5-fluorouracil, N-(2′-furanidyl)-5-fluorouracil, DaiichiSeiyaku FO-152, isopropyl pyrrolizine, Lilly LY-188011, Lilly LY-264618,methobenzaprim, methotrexate, Wellcome MZPES, norspermidine, NCINSC-127716, NCI NSC-264880, NCI NSC-39661, NCI NSC-612567,Warner-Lambert PALA, pentostatin, piritrexim, plicamycin, Asahi ChemicalPL-AC, Takeda TAC-788, thioguanine, tiazofurin, Erbamont TIF,trimetrexate, tyrosine kinase inhibitors, tyrosine protein kinaseinhibitors, Taiho UFT, uricytin, Shionogi 254-S, aldo-phosphamideanalogues, altretamine, anaxirone, Boehringer Mannheim BBR-2207,bestrabucil, budotitane, Wakunaga CA-102, carboplatin, carmustine,Chinoin-139, Chinoin-153, chlorambucil, cisplatin, cyclophosphamide,American Cyanamid CL-286558, Sanofi CY-233, cyplatate, Degussa D-19-384,Sumimoto DACHP(Myr)2, diphenylspiromustine, diplatinum cytostatic, Erbadistamycin derivatives, Chugai DWA-2114R, ITI E09, elmustine, ErbamontFCE-24517, estramustine phosphate sodium, fotemustine, Unimed G-6-M,Chinoin GYKI-17230, hepsul-fam, ifosfamide, iproplatin, lomustine,mafosfamide, mitolactol, Nippon Kayaku NK-121, NCI NSC-264395, NCINSC-342215, oxaliplatin, Upjohn PCNU, prednimustine, Proter PTT-119,ranimustine, semustine, SmithKline SK&F-101772, Yakult Honsha SN-22,spiromus-tine, Tanabe Seiyaku TA-077, tauromustine, temozolomide,teroxirone, tetraplatin, trimelamol, Taiho 4181-A, aclarubicin,actinomycin D, actinoplanone, Erbamont ADR-456, aeroplysinin derivative,Ajinomoto AN-201-II, Ajinomoto AN-3, Nippon Soda anisomycins,anthracycline, azino-mycin-A, bisucaberin, Bristol-Myers BL-6859,Bristol-Myers BMY-25067, Bristol-Myers BMY-25551, Bristol-MyersBMY-26605, Bristol-Myers BMY-27557, Bristol-Myers BMY-28438, bleomycinsulfate, bryostatin-1, Taiho C-1027, calichemycin, chromoximycin,dactinomycin, daunorubicin, Kyowa Hakko DC-102, Kyowa Hakko DC-79, KyowaHakko DC-88A, Kyowa Hakko DC89-A1, Kyowa Hakko DC92-B, ditrisarubicin B,Shionogi DOB-41, doxorubicin, doxorubicin-fibrinogen, elsamicin-A,epirubicin, erbstatin, esorubicin, esperamicin-A1, esperamicin-A1b,Erbamont FCE-21954, Fujisawa FK-973, fostriecin, Fujisawa FR-900482,glidobactin, gregatin-A, grincamycin, herbimycin, idarubicin, illudins,kazusamycin, kesarirhodins, Kyowa Hakko KM-5539, Kirin Brewery KRN-8602,Kyowa Hakko KT-5432, Kyowa Hakko KT-5594, Kyowa Hakko KT-6149, AmericanCyanamid LL-D49194, Meiji Seika ME 2303, menogaril, mitomycin,mitoxantrone, SmithKline M-TAG, neoenactin, Nippon Kayaku NK-313, NipponKayaku NKT-01, SRI International NSC-357704, oxalysine, oxaunomycin,peplomycin, pilatin, pirarubicin, porothramycin, pyrindamycin A, TobishiRA-I, rapamycin, rhizoxin, rodorubicin, sibanomicin, siwenmycin,Sumitomo SM-5887, Snow Brand SN-706, Snow Brand SN-07, sorangicin-A,sparsomycin, SS Pharmaceutical SS-21020, SS Pharmaceutical SS-7313B, SSPharmaceutical SS-9816B, steffimycin B, Taiho 4181-2, talisomycin,Takeda TAN-868A, terpentecin, thrazine, tricrozarin A, Upjohn U-73975,Kyowa Hakko UCN-10028A, Fujisawa WF-3405, Yoshitomi Y-25024 zorubicin,alpha-carotene, alpha-difluoromethyl-arginine, acitretin, Biotec AD-5,Kyorin AHC-52, alstonine, amonafide, amphethinile, amsacrine, Angiostat,ankinomycin, anti-neoplaston A10, antineoplaston A2, antineoplaston A3,antineoplaston A5, antineoplaston AS2-1, Henkel APD, aphidicolinglycinate, asparaginase, Avarol, baccharin, batracylin, benfluron,benzotript, Ipsen-Beaufour BIM-23015, bisantrene, Bristo-MyersBMY-40481, Vestar boron-10, bromofosfamide, Wellcome BW-502, WellcomeBW-773, caracemide, carmethizole hydrochloride, Ajinomoto CDAF,chlorsulfaquinoxalone, Chemex CHX-2053, Chemex CHX-100, Warner-LambertCI-921, Warner-Lambert CI-937, Warner-Lambert CI-941, Warner-LambertCI-958, clanfenur, claviridenone, ICN compound 1259, ICN compound 4711,Contracan, Yakult Honsha CPT-11, crisnatol, curaderm, cytochalasin B,cytarabine, cytocytin, Merz D-609, DABIS maleate, dacarbazine,datelliptinium, didemnin-B, dihaematoporphyrin ether, dihydrolenperone,dinaline, distamycin, Toyo Pharmar DM-341, Toyo Pharmar DM-75, DaiichiSeiyaku DN-9693, elliprabin, elliptinium acetate, Tsumura EPMTC,ergotamine, etoposide, etretinate, fenretinide, Fujisawa FR-57704,gallium nitrate, genkwadaphnin, Chugai GLA-43, Glaxo GR-63178, grifolanNMF-SN, hexadecylphosphocholine, Green Cross HO-221, homoharringtonine,hydroxyurea, BTG ICRF-187, ilmofosine, isoglutamine, isotretinoin,Otsuka JI-36, Ramot K-477, Otsuak K-76COONa, Kureha Chemical K-AM, MECTCorp KI-8110, American Cyanamid L-623, leukoregulin, lonidamine,Lundbeck LU-23-112, Lilly LY-186641, NCI (US) MAP, marycin, Merrel DowMDL-27048, Medco MEDR-340, merbarone, merocyanine derivatives,methylanilinoacridine, Molecular Genetics MGI-136, minactivin,mitonafide, mitoquidone, mopidamol, motretinide, Zenyaku Kogyo MST-16,N-(retinoyl)amino acids, Nisshin Flour Milling N-021,N-acylated-dehydroalanines, nafazatrom, Taisho NCU-190, nocodazolederivative, Normosang, NCI NSC-145813, NCI NSC-361456, NCI NSC-604782,NCI NSC-95580, octreotide, Ono ONO-112, oquizanocine, Akzo Org-10172,pancratistatin, pazelliptine, Warner-Lambert PD-111707, Warner-LambertPD-115934, Warner-Lambert PD-131141, Pierre Fabre PE-1001, ICRT peptideD, piroxantrone, polyhaematoporphyrin, polypreic acid, Efamol porphyrin,probimane, procarbazine, proglumide, Invitron protease nexin I, TobishiRA-700, razoxane, Sapporo Breweries RBS, restrictin-P, retelliptine,retinoic acid, Rhone-Poulenc RP-49532, Rhone-Poulenc RP-56976,SmithKline SK&F-104864, Sumitomo SM-108, Kuraray SMANCS, SeaPharmSP-10094, spatol, spirocyclopropane derivatives, spirogermanium, Unimed,SS Pharmaceutical SS-554, strypoldinone, Stypoldione, Suntory SUN 0237,Suntory SUN 2071, superoxide dismutase, Toyama T-506, Toyama T-680,taxol, Teijin TEI-0303, teniposide, thaliblastine, Eastman Kodak TJB-29,tocotrienol, Topostin, Teijin TT-82, Kyowa Hakko UCN-01, Kyowa HakkoUCN-1028, ukrain, Eastman Kodak USB-006, vinblastine sulfate,vincristine, vindesine, vinestramide, vinorelbine, vintriptol,vinzolidine, withanolides, Yamanouchi YM-534, uroguanylin,combretastatin, dolastatin, idarubicin, epirubicin, estramustine,cyclophosphamide, 9-amino-2-(S)-camptothecin, topotecan, irinotecan(Camptosar), exemestane, decapeptyl (tryptorelin), or an omega-3 fattyacid.

Examples of radioprotective agents which may be used in a combinationtherapy with the compounds of this invention include AD-5, adchnon,amifostine analogues, detox, dimesna, 1-102, MM-159,N-acylated-dehydroalanines, TGF-Genentech, tiprotimod, amifostine,WR-151327, FUT-187, ketoprofen transdermal, nabumetone, superoxidedismutase (Chiron) and superoxide dismutase Enzon.

The compounds of the present invention will also be useful in treatmentor prevention of angiogenesis-related disorders or conditions, forexample, tumor growth, metastasis, macular degeneration, andatherosclerosis.

In a further embodiment, the present invention also provides therapeuticcombinations for the treatment or prevention of ophthalmic disorders orconditions such as glaucoma. For example the present inventive compoundsadvantageously will be used in therapeutic combination with a drug whichreduces the intraocular pressure of patients afflicted with glaucoma.Such intraocular pressure-reducing drugs include without limitationlatanoprost, travoprost, bimatoprost, or unoprostol. The therapeuticcombination of a compound of the present invention plus an intraocularpressure-reducing drug will be useful because each is believed toachieve its effects by affecting a different mechanism.

In another combination of the present invention, the present inventivecompounds can be used in therapeutic combination with anantihyperlipidemic or cholesterol-lowering drug such as a benzothiepineor a benzothiazepine antihyperlipidemic drug. Examples of benzothiepineantihyperlipidemic drugs useful in the present inventive therapeuticcombination can be found in U.S. Pat. No. 5,994,391, herein incorporatedby reference. Some benzothiazepine antihyperlipidemic drugs aredescribed in WO 93/16055. Alternatively, the antihyperlipidemic orcholesterol-lowering drug useful in combination with a compound of thepresent invention can be an HMG Co-A reductase inhibitor. Examples ofHMG Co-A reductase inhibitors useful in the present therapeuticcombination include, individually, benfluorex, fluvastatin, lovastatin,provastatin, simvastatin, atorvastatin, cerivastatin, bervastatin,ZD-9720 (described in PCT Patent Application No. WO 97/06802), ZD-4522(CAS No. 147098-20-2 for the calcium salt; CAS No. 147098-18-8 for thesodium salt; described in European Patent No. EP 521471), BMS 180431(CAS No. 129829-03-4), or NK-104 (CAS No. 141750-63-2). The therapeuticcombination of a compound of the present invention plus anantihyperlipidemic or cholesterol-lowering drug will be useful, forexample, in reducing the risk of formation of atherosclerotic lesions inblood vessels. For example, atherosclerotic lesions often initiate atinflamed sites in blood vessels. It is established thatantihyperlipidemic or cholesterol-lowering drug reduce risk of formationof atherosclerotic lesions by lowering lipid levels in blood. Withoutlimiting the invention to a single mechanism of action, it is believedthat one way the compounds of the present combination will work inconcert to provide improved control of atherosclerotic lesions by, forexample, reducing inflammation of the blood vessels in concert withlowering blood lipid levels.

In another embodiment of the invention, the present compounds can beused in combination with other compounds or therapies for the treatmentof central nervous conditions or disorders such as migraine. Forexample, the present compounds can be used in therapeutic combinationwith caffeine, a 5-HT-1B/1D agonist (for example, a triptan such assumatriptan, naratriptan, zolmitriptan, rizatriptan, almotriptan, orfrovatriptan), a dopamine D4 antagonist (e.g., sonepiprazole), aspirin,acetaminophen, ibuprofen, indomethacin, naproxen sodium, isometheptene,dichloralphenazone, butalbital, an ergot alkaloid (e.g., ergotamine,dihydroergotamine, bromocriptine, ergonovine, or methyl ergonovine), atricyclic antidepressant (e.g., amitriptyline or nortriptyline), aserotonergic antagonist (e.g., methysergide or cyproheptadine), abeta-andrenergic antagonist (e.g., propranolol, timolol, atenolol,nadolol, or metprolol), or a monoamine oxidase inhbitor (e.g.,phenelzine or isocarboxazid).

A further embodiment provides a therapeutic combination of a compound ofthe present invention with an opioid compound. Opioid compounds usefulin this combination include without limitation morphine, methadone,hydromorphone, oxymorphone, levorphanol, levallorphan, codeine,dihydrocodeine, dihydrohydroxycodeinone, pentazocine, hydrocodone,oxycodone, nalmefene, etorphine, levorphanol, fentanyl, sufentanil,DAMGO, butorphanol, buprenorphine, naloxone, naltrexone, CTOP,diprenorphine, beta-funaltrexamine, naloxonazine, nalorphine,pentazocine, nalbuphine, naloxone benzoylhydrazone, bremazocine,ethylketocyclazocine, U50,488, U69,593, spiradoline,nor-binaltorphimine, naltrindole, DPDPE, [D-la², glu⁴]deltorphin, DSLET,met-enkephalin, leu-enkaphalin, beta-endorphin, dynorphin A, dynorphinB, and alpha-neoendorphin. An advantage to the combination of thepresent invention with an opioid compound is that the present inventivecompounds will allow a reduction in the dose of the opioid compound,thereby reducing the risk or severity of opioid side effects, such asopioid addiction.

The term “alkyl”, alone or in combination, means an acyclic alkylradical, linear or branched, preferably containing from 1 to about 10carbon atoms and more preferably containing from 1 to about 6 carbonatoms. “Alkyl” also encompasses cyclic alkyl radicals containing from 3to about 7 carbon atoms, preferably from 3 to 5 carbon atoms. Said alkylradicals can be optionally substituted with groups as defined below.Examples of such radicals include methyl, ethyl, chloroethyl,hydroxyethyl, n-propyl, isopropyl, n-butyl, cyanobutyl, isobutyl,sec-butyl, tert-butyl, pentyl, aminopentyl, iso-amyl, hexyl, octyl andthe like.

The term “alkenyl” refers to an unsaturated, acyclic hydrocarbonradical, linear or branched, in so much as it contains at least onedouble bond. Such radicals containing from 2 to about 6 carbon atoms,preferably from 2 to about 4 carbon atoms, more preferably from 2 toabout 3 carbon atoms. Said alkenyl radicals may be optionallysubstituted with groups as defined below. Examples of suitable alkenylradicals include propenyl, 2-chloropropylenyl, buten-1-yl, isobutenyl,penten-1-yl, 2-methylbuten-1-yl, 3-methylbuten-1-yl, hexen-1-yl,3-hydroxyhexen-1-yl, hepten-1-yl, and octen-1-yl, and the like.

The term “alkynyl” refers to an unsaturated, acyclic hydrocarbonradical, linear or branched, in so much as it contains one or moretriple bonds, such radicals containing 2 to about 6 carbon atoms,preferably from 2 to about 4 carbon atoms, more preferably from 2 toabout 3 carbon atoms. Said alkynyl radicals may be optionallysubstituted with groups as defined below. Examples of suitable alkynylradicals include ethynyl, propynyl, hydroxypropynyl, butyn-1-yl,butyn-2-yl, pentyn-1-yl, pentyn-2-yl, 4-methoxypentyn-2-yl,3-methylbutyn-1-yl, hexyn-1-yl, hexyn-2-yl, hexyn-3-yl,3,3-dimethylbutyn-1-yl radicals and the like.

The term “alkoxy” embrace linear or branched oxy-containing radicalseach having alkyl portions of 1 to about 6 carbon atoms, preferably 1 toabout 3 carbon atoms, such as a methoxy radical. The term “alkoxyalkyl”also embraces alkyl radicals having one or more alkoxy radicals attachedto the alkyl radical, that is, to form monoalkoxyalkyl and dialkoxyalkylradicals. Examples of such radicals include methoxy, ethoxy, propoxy,butoxy and tert-butoxy alkyls. The “alkoxy” radicals may be furthersubstituted with one or more halo atoms, such as fluoro, chloro orbromo, to provide “haloalkoxy” radicals. Examples of such radicalsinclude fluoromethoxy, chloromethoxy, trifluoromethoxy, difluoromethoxy,trifluoroethoxy, fluoroethoxy, tetrafluoroethoxy, pentafluoroethoxy, andfluoropropoxy.

The term “alkylthio” embraces radicals containing a linear or branchedalkyl radical, of 1 to about 6 carbon atoms, attached to a divalentsulfur atom. An example of “lower alkylthio” is methylthio (CH₃—S—).

The term “alkylthioalkyl” embraces alkylthio radicals, attached to analkyl group. Examples of such radicals include methylthiomethyl.

The term “halo” means halogens such as fluorine, chlorine, bromine oriodine atoms.

The term “heterocyclyl” means a saturated or unsaturated mono- ormulti-ring carbocycle wherein one or more carbon atoms is replaced by N,S, P, or O. This includes, for example, the following structures:

wherein Z, Z¹, Z² or Z³ is C, S, P, O, or N, with the proviso that oneof Z, Z¹, Z² or Z³ is other than carbon, but is not O or S when attachedto another Z atom by a double bond or when attached to another O or Satom. Furthermore, the optional substituents are understood to beattached to Z, Z¹, Z² or Z³ only when each is C. The term “heterocyclyl”also includes fully saturated ring structures such as piperazinyl,dioxanyl, tetrahydrofuranyl, oxiranyl, aziridinyl, morpholinyl,pyrrolidinyl, piperidinyl, thiazolidinyl, and others. The term“heterocyclyl” also includes partially unsaturated ring structures suchas dihydrofuranyl, pyrazolinyl, imidazolinyl, pyrrolinyl, chromanyl,dihydrothiophenyl, and others.

The term “heteroaryl” means a fully unsaturated heterocycle.

In either “heterocycle” or “heteroaryl,” the point of attachment to themolecule of interest can be at the heteroatom or elsewhere within thering.

The term “cycloalkyl” means a mono- or multi-ringed carbocycle whereineach ring contains three to about seven carbon atoms, preferably threeto about five carbon atoms.

Examples include radicals such as cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl, cycloalkenyl, and cycloheptyl. The term “cycloalkyl”additionally encompasses spiro systems wherein the cycloalkyl ring has acarbon ring atom in common with the seven-membered heterocyclic ring ofthe benzothiepine.

The term “oxo” means a doubly bonded oxygen.

The term “alkoxy” means a radical comprising an alkyl radical that isbonded to an oxygen atom, such as a methoxy radical. More preferredalkoxy radicals are “lower alkoxy” radicals having one to about tencarbon atoms. Still more preferred alkoxy radicals have one to about sixcarbon atoms. Examples of such radicals include methoxy, ethoxy,propoxy, isopropoxy, butoxy and tert-butoxy.

The term “aryl” means a fully unsaturated mono- or multi-ringcarbocycle, including, but not limited to, substituted or unsubstitutedphenyl, naphthyl, or anthracenyl.

The term “Combination therapy” means the administration of two or moretherapeutic agents to treat a therapeutic condition or disorderdescribed in the present disclosure, for example atherosclerosis, pain,inflammation, migraine, neoplasia, angiogenisis-related condition ordisorder, or other. Such administration encompasses co-administration ofthese therapeutic agents in a substantially simultaneous manner, such asin a single capsule having a fixed ratio of active ingredients or inmultiple, separate capsules for each active ingredient. In addition,such administration also encompasses use of each type of therapeuticagent in a sequential manner. In either case, the treatment regimen willprovide beneficial effects of the drug combination in treating theconditions or disorders described herein.

The phrase “therapeutically effective” is intended to qualify thecombined amount of active ingredients in the combination therapy. Thiscombined amount will achieve the goal of reducing or eliminating thehyperlipidemic condition.

In one embodiment, the present invention provides a compound or a saltthereof, the compound having a structure corresponding to Formula 1:

In the structure of Formula 1, X is selected from the group consistingof —S—, —S(O)—, and —S(O)₂—. Preferably, X is —S—. R² is selected fromthe group consisting of C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₅alkoxy-C₁ alkyl, and C₁-C₅ alkylthio-C₁ alkyl wherein each of thesegroups is optionally substituted by one or more substituent selectedfrom the group consisting of —OH, alkoxy, and halogen. Preferably, R² isC₁-C₆ alkyl optionally substituted with a substituent selected from thegroup consisting of —OH, alkoxy, and halogen. With respect to R³ and R⁸,R⁸ is selected from the group consisting of —OR¹⁴ and —N(R¹⁵)(R¹⁶), andR³ is selected from the group consisting of —H, —OH, —C(O)—R¹⁷,—C(O)—O—R¹⁸, and —C(O)—S—R¹⁹; or R¹⁹ is —N(R²⁰)—, and R³ is —C(O)—,wherein R⁸ and R³ together with the atoms to which they are attachedform a ring; or R⁸ is —O—, and R³ is —C(R²¹)(R²²)—, wherein R⁸ and R³together with the atoms to which they are attached form a ring. If R³ is—C(R²¹)(R²²)—, then R⁴ is —C(O)—O—R²³; otherwise R⁴ is —H. R¹, R⁵, R⁶,and R⁷ independently are selected from the group consisting of —H,halogen, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, and C₁-C₅alkoxy-C₁alkyl. R⁹ and R¹⁰ independently are selected from the groupconsisting of —H, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, and C₁-C₅alkoxy-C₁ alkyl. With respect to R¹¹ and R¹², R¹¹ is selected from thegroup consisting of —H, —OH, —C(O)—O—R²⁴, and —C(O)—S—R²⁵, and R¹² isselected from the group consisting of —H, —OH, —C(O)—O—R²⁶, and—C(O)—S—R²⁷; or R¹¹ is —O—, and R¹² is —C(O)—, wherein R¹¹ and R¹²together with the atoms to which they are attached form a ring; or R¹¹is —C(O)—, and R¹² is —O—, wherein R¹¹ and R¹² together with the atomsto which they are attached form a ring. R¹³ is C₁ alkyl. R¹⁴ is selectedfrom the group consisting of —H and C₁-C₆ alkyl, wherein when R¹⁴ isC₁-C₆ alkyl, R¹⁴ is optionally substituted by one or more moietiesselected from the group consisting of cycloalkyl, heterocyclyl, aryl,and heteroaryl. With respect to R¹⁵ and R¹⁶, R¹⁵ is selected from thegroup consisting of —H, alkyl, and alkoxy, and R¹⁶ is selected from thegroup consisting of —H, —OH, alkyl, alkoxy, —C(O)—R^(27a), —C(O)—O—R²⁸,and —C(O)—S—R²⁹; wherein when R¹⁵ and R¹⁶ independently are alkyl oralkoxy, R¹⁵ and R¹⁶ independently are optionally substituted with one ormore moieties selected from the group consisting of cycloalkyl,heterocyclyl, aryl, and heteroaryl; or R¹⁵ is —H; and R¹⁶ is selectedfrom the group consisting of cycloalkyl, heterocyclyl, aryl, andheteroaryl. R¹⁷, R¹⁸, R¹⁹, R²⁰, R²¹, R²², R²³, R²⁴, R²⁵, R²⁶, R²⁷,R^(27a), R²⁸, and R²⁹ independently are selected from the groupconsisting of —H and alkyl, wherein alkyl is optionally substituted byone or more moieties selected from the group consisting of cycloalkyl,heterocyclyl, aryl, and heteroaryl. When any of R¹, R², R³, R⁴, R⁵, R⁶,R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³, R¹⁴, R¹⁵, R¹⁶, R¹⁷, R¹⁸, R¹⁹, R²⁰, R²¹,R²², R²³, R²⁴, R²⁵, R²⁶, R²⁷, R^(27a), R²⁸, and R²⁹ independently is amoiety selected from the group consisting of alkyl, alkenyl, alkynyl,alkoxy, alkylthio, cycloalkyl, heterocyclyl, aryl, and heteroaryl, thenthe moiety is optionally substituted by one or more substituent selectedfrom the group consisting of —OH, alkoxy, and halogen.

In a preferred embodiment, R⁸ is —OH. When R⁸ is —OH, preferably X is S.In a further embodiment, R¹, R⁵, R⁶, R⁷, R⁹, and R¹⁰ independently areselected from the group consisting of —H and C₁-C₃ alkyl. Preferably R⁵,R⁶, R⁷, R⁹, R¹⁰ each are —H. R¹³ can be a variety of groups, for examplefluoromethyl or methyl. R¹ can be C₁-C₆ alkyl optionally substitutedwith a substituent selected from the group consisting of —OH andhalogen; preferably R¹ is C₁ alkyl optionally substituted with halogen;more preferably R¹ is selected from the group consisting offluoromethyl, hydroxymethyl, and methyl. In one important embodiment R¹can be methyl. Alternatively, R¹ can be fluoromethyl. In anotheralternative R¹ can be hydroxymethyl. In another embodiment, R² is C₁-C₆alkyl optionally substituted with a substituent selected from the groupconsisting of —OH, alkoxy, and halogen. In one preferred embodiment R²is C₁ alkyl optionally substituted with halogen. For example, R² can bemethyl. Alternatively, R² can be fluoromethyl. In yet another example,R² can be hydroxymethyl. In still another example, R² can bemethoxymethyl.

In the compounds of the present invention, it is preferred that R³, R⁴,R¹¹ and R¹² each is —H. In this embodiment, it is further preferred thatR¹, R⁵, R⁶, R⁷, R⁹, and R¹⁰ independently are selected from the groupconsisting of —H and C₁-C₃ alkyl. Preferably R⁵, R⁶, R⁷, R⁹, R¹⁰ each is—H. In this further embodiment, R¹³ can be, for example, fluoromethyl,or in another example R¹³ can be methyl. In preferred compounds of theseexamples, R² is C₁-C₆ alkyl optionally substituted with a substituentselected from the group consisting of —OH, alkoxy, and halogen.Preferably R² is C₁ alkyl optionally substituted with halogen. In onesuch example R² is fluoromethyl. In another example R² is methyl.Alternatively R² can be hydroxymethyl. In another alternative, R² can bemethoxymethyl.

When R¹³ is methyl, R¹ can be, for example, —H or C₁-C₆ alkyl optionallysubstituted with a substituent selected from the group consisting of —OHand halogen. In a preferred embodiment R¹ is —H. Alternatively, R¹ canbe C₁-C₆ alkyl optionally substituted with a substituent selected fromthe group consisting of —OH and halogen. For example R¹ can be methyl,ethyl, n-propyl, i-propyl, n-butyl, sec-butyl, isobutyl, t-butyl, apentyl isomer, or a hexyl isomer. For example, R¹ can be ethyl.Alternatively, R¹ can be C₁ alkyl optionally substituted with asubstituent selected from the group consisting of —OH and halogen; forexample R¹ can be methyl. Alternatively, R¹ can be fluoromethyl. Inanother alternative, R¹ can be hydroxymethyl.

In another embodiment R⁸ can be —OR¹⁴. R¹⁴ can be as defined above.Preferably R¹⁴ is C₁-C₆ alkyl optionally substituted by one or moremoieties selected from the group consisting of cycloalkyl, heterocyclyl,aryl, and heteroaryl; more preferably R¹⁴ is C₁-C₃ alkyl; and morepreferably still R¹⁴ is methyl. In yet another embodiment of compound 1,R⁸ can be —N(R¹⁵)(R¹⁶), wherein R¹⁵ and R¹⁶ are as defined above. Instill another embodiment R⁸ can be —N(R²⁰)—, and R³ can be —C(O)—,wherein R⁸ and R³ together with the atoms to which they are attachedform a ring. In another embodiment still, R⁸ can be —O—, and R³ can be—C(R²¹)(R²²)—, wherein R⁸ and R³ together with the atoms to which theyare attached form a ring.

In the compound of Formula 1, R¹¹ can be selected from the groupconsisting of —OH, —C(O)—O—R²⁴, and —C(O)—S—R²⁵. Preferably R¹¹ is —OH.In a further embodiment R¹² is —H when R¹¹ is —OH.

However, the present invention also provides useful compounds of Formula1 in which R¹¹ is —O—, and R¹² is —C(O)—, wherein R¹¹ and R¹² togetherwith the atoms to which they are attached form a ring. In another usefulembodiment R¹¹ is —C(O)—, and R¹² is —O—, wherein R¹¹ and R¹² togetherwith the atoms to which they are attached form a ring. Alternatively,R¹² can be selected from the group consisting of —OH, —C(O)—O—R²⁶, and—C(O)—S—R²⁷. In this alternative, R¹¹ is preferably —H.

The present invention also provides pharmaceutically-acceptable salts ofthe compounds of Formula 1. For example, such a pharmaceuticallyacceptable salt can be one in which the present inventive compound is ina cationic form with at least one anionic counterion. Examples ofanionic counterions useful in the pharmaceutically-acceptable salts ofthe present invention include a halide, a carboxylate, a sulfonate, asulfate, a phosphate, a phosphonate, a resin-bound anion, or a nitrate.When the anionic counterion is a halide, it can be, for examplefluoride, chloride, bromide, or iodide. Preferably the halide counterionis chloride. When the anionic counterion is a carboxylate (i.e., theanionic form of a compound containing a carboxylic acid functionalgroup), the carboxylate counterion can vary widely. The carboxylatecounterion can be, for example, formate, acetate, propionate,trifluoroacetate, succinate, salicylate, DL-aspartate, D-aspartate,L-aspartate, DL-glutamate, D-glutamate, L-glutamate, glycerate,succinate, steric, DL-tartarate, D-tartarate, L-tartarate,(±)-mandelate, (R)-(−)-mandelate, (S)-(+)-mandelate, citrate, mucate,maleate, malonate, benzoate, DL-malate, D-malate, L-malate, hemi-malate,1-adamantaneacetate, 1-adamantanecarboxylate, flavianate,sulfonoacetate, (±)-lactate, L-(+)-lactate, D-(−)-lactate, pamoate,D-alpha-galacturonate, glycerate, DL-ascorbate, D-ascorbate,L-ascorbate, DL-cystate, D-cystate, L-cystate, DL-homocystate,D-homocystate, L-homocystate, DL-cysteate, D-cysteate, L-cysteate,(4S)-hydroxy-L-proline, cyclopropane-1,1-dicarboxylate,2,2-dimethylmalonate, squarate, tyrosine anion, proline anion, fumarate,1-hydroxy-2-naphthoate, phosphonoacetate, carbonate, bicarbonate,3-phosphonopropionate, DL-pyroglutamate, D-pyroglutamate, orL-pyroglutamate. Alternatively, the anionic counterion can be asulfonate. For example the sulfonate counterion can be methanesulfonate,toluenesulfonate, benzenesulfonate, trifluoromethylsulfonate,ethanesulfonate, (±)-camphorsulfonate, naphthalenesulfonate,1R-(−)-camphorsulfonate, 1S-(+)-camphorsulfonate, 2-mesitylenesulfonate,1,5-naphthalenedisulfonate, 1,2-ethanedisulfonate,1,3-propanedisulfonate, 3-(N-morpholino)propane sulfonate,biphenylsulfonate, isethionate, or 1-hydroxy-2-naphthalenesulfonate. Inanother embodiment the anionic counterion can be a sulfate. Examples ofsulfates useful in the present invention include without limitationsulfate, monopotassium sulfate, monosodium sulfate, and hydrogensulfate. The anionic counterion can be a sulfamate. When the anioniccounterion is a phosphate, it can be, for example, phosphate, dihydrogenphosphate, potassium hydrogen phosphate, dipotassium phosphate,potassium phosphate, sodium hydrogen phosphate, disodium phosphate,sodium phosphate, calcium dihydrogen phosphate, calcium phosphate,calcium hydrogen phosphate, calcium phosphate tribasic, orhexafluorophosphate. The anionic counterion can be a phosphonate. Forexample, the phosphonate counterion can be vinylphosphonate,2-carboxyethylphosphonate or phenylphosphonate. Alternatively, theanionic counterion can be nitrate. The salt can also result from theaddition of the compound with an oxide such as zinc oxide.

The anionic counterion can, if desired, be bound to a polymeric resin.In other words, the anionic counterion can be a resin-bound anion. Forexample the resin-bound anion can be a polyacrylate resin wherein theresin contains anionic carboxylate groups. An example of a polyacrylateresin useful in the salts of the present invention is Bio-Rex 70(produced by Bio-Rad). In an alternative example, the resin-bound anioncan be a sulfonated poly (styrene divinylbenzene) copolymer resin.Non-limiting examples of sulfonated poly (styrene divinylbenzene)copolymer resins useful as anionic counterions in the present inventioninclude Amberlite IPR-69 (Rohm & Haas) or Dowex 50WX4-400 (Dow). Thepolyacrylate resin or the sulfonated poly(styrene divinylbenzene) resincan be, if desired, crosslinked with a crosslinking agent such asdivinylbenzene.

In another embodiment, the pharmaceutically acceptable salt of thecompound of Formula 1 can be one in which the present inventive compoundis in an anionic form with at least one cationic counterion. Thecationic counterion can be, for example, an ammonium cation, a alkalimetal cation, an alkaline earth metal cation, a transition metal cation,or a resin-bound cation. When the cationic counterion is an ammoniumcation, it can be substituted or unsubstituted. For example, theammonium cation can be an alkylammonium cation or a di-, tri-, ortetra-alkylammonium cation. Alternatively the ammonium cation can be anarylammonium or a di-, tri-, or tetra-arylammonium cation. The ammoniumcation can contain both alkyl and aryl groups. The ammonium cation canbe aromatic, for example a pyridinium cation. Other functional groupscan also be present in the ammonium cation. The ammonium cation can be,for example, ammonium, methyl ammonium, dimethylammonium,trimethylammonium, tetramethylammonium, ethanolammonium,dicyclohexylammonium, guanidinium, or ethylenediammonium cation.Alternatively the cationic counterion can be an alkali metal cation suchas lithium cation, sodium cation, potassium cation or cesium cation. Inanother alternative the cationic counterion can be an alkaline earthmetal cation such as beryllium cation, magnesium cation, or calciumcation. The cation, if preferred, can be a transition metal cation suchas zinc cation.

The cationic counterion can, if desired, be bound to a polymeric resin.In other words, the anionic counterion can be a resin-bound cation. Forexample, the resin-bound cation can be a cationically functionalizedpoly(styrene divinylbenzene) resin. An example of a cationicallyfunctionalized poly(styrene divinylbenzene) resin useful in the presentinvention is Bio-Rex-5 (Bio-Rad), an ammonium-functionalized resin. Inanother alternative, the resin-bound cation can be a cationicallyfunctionalized polyacrylic resin such as an aminated polyacrylic resin.An example of an aminated polyacrylic resin useful as the cationiccounterion of the present invention is AG-4-XR (Bio-Rad).

In yet another embodiment of the present invention the compound ofFormula 1 can be present in a zwiltterionic form. In other words, thecompound can contain both cationic and anionic sites within themolecule. Such a zwitterionic form can exist without a separatecounterion or it can exist with both a cationic counterion and ananionic counterion.

Another embodiment provides a method for the treatment or prevention ofan inflammation-related disorder wherein the method comprises treating asubject in need thereof with an inflammation-related disorder-treatingor preventing amount of a compound or salt of the present invention.

In yet another embodiment the present invention provides a method forthe preparation of a compound of Formula 21:

or a salt thereof, wherein: X is selected from the group consisting of—S—, —S(O)—, and —S(O)₂—; R² is selected from the group consisting ofC₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₅ alkoxy-C₁ alkyl, andC₁-C₅ alkylthio-C₁ alkyl; R³⁰ is selected from the group consisting of—H, —OH, —C(O)—R¹⁷, —C(O)—O—R¹⁸, and —C(O)—S—R¹⁹; R¹, R⁵, R⁶, and R⁷independently are selected from the group consisting of —H, halogen,C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, and C₁-C₅ alkoxy-C₁ alkyl; R⁹and R¹⁰ independently are selected from the group consisting of —H,C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, and C₁-C₅ alkoxy-C₁ alkyl;with respect to R¹¹ and R¹²:

-   -   R¹¹ is selected from the group consisting of —H, —OH,        —C(O)—O—R²⁴, and —C(O)—S—R²⁵; and R¹² is selected from the group        consisting of —H, —OH, —C(O)—O—R²⁶, and —C(O)—S—R²⁷; or R¹¹ is        —O—, and R¹² is —C(O)—, wherein R¹¹ and R¹² together with the        atoms to which they are attached form a ring; or R¹¹ is —C(O)—,        and R¹² is —O—, wherein R¹¹ and R¹² together with the atoms to        which they are attached form a ring;    -   R¹³ is C₁ alkyl;    -   R¹⁷, R¹⁸, R¹⁹, R²⁴, R²⁵, R²⁶, R²⁷, and R^(27a) independently are        selected from the group consisting of —H and alkyl, which is        optionally substituted by one or more moieties selected from the        group consisting of cycloalkyl, heterocyclyl, aryl, and        heteroaryl; and when any of R¹, R², R⁴, R⁵, R⁶, R⁷, R⁹, R¹⁰,        R¹¹, R¹², R¹³, R¹⁴, R¹⁵, R¹⁶, R¹⁷, R¹⁸, R¹⁹, R²⁴, R²⁵, R²⁶, R²⁷,        and R^(27a) independently is a moiety selected from the group        consisting of alkyl, alkenyl, alkynyl, alkoxy, alkylthio,        cycloalkyl, heterocyclyl, aryl, and heteroaryl, then the moiety        is optionally substituted by one or more substituent selected        from the group consisting of —OH, alkoxy, and halogen. The        present method of preparing compound 21 comprises treating a        diamine compound having a structure corresponding to Formula 22:        (or a salt thereof) with an alkyl acetimidate having a structure        corresponding to Formula 23:        (or a salt thereof) wherein R³¹ is C₁-C₆ alkyl. Preferably R¹¹        is selected from the group consisting of —H and —OH. R¹¹ can,        for example be —H. Alternatively, R¹¹ can be —OH. When R¹¹ is —H        or —OH, preferably R¹³ is methyl or halomethyl. More preferably        R¹³ is methyl. Also when R¹¹ is —H or —OH, preferably R³¹ is        C₁-C₃ alkyl, and more preferably ethyl. In a preferred        embodiment, the treating of the diamine compound with the alkyl        acetimidate is performed in the presence of a base. For example,        the base can be a hydrazine, a metal sulfide, a metal hydroxide,        a metal alkoxide, an amine, a hydroxylamine, a metal hydride, a        metal amide complex, or a basic resin. When the base is a basic        resin it can be, for example a polymer-bound        diazabicyclo[4.4.0]dec-2-ene resin. For example, the basic resin        can have a poly(styrene divinylbenzene) copolymer backbone with        diazabicyclo[4.4.0]dec-2-ene bonded to the copolymer. When the        base is an amine, it can be essentially any substituted or        unsubstituted amine. For example, the amine can be        1,5-diazabicyclo[4.3.0]non-5-ene; 1,4-diazabicyclo[2.2.2]octane;        or 1,8-diazabicyclo[5.4.0]undec-7-ene. When the base is an        alkali metal hydroxide it can be, for example, potassium        hydroxide or sodium hydroxide. When the base is a metal hydride        it can be, for example, sodium hydride, potassium hydride, or        calcium hydride.

In a further embodiment of the present invention provides a method forthe preparation of a diamine compound having a structure correspondingto Formula 22 (or a salt thereof), wherein the method comprises treatinga protected diamine compound having the structure corresponding toFormula 24:

(or a salt thereof) wherein R³³ is selected from the group consisting of—NH₂ and a protected amino group; and R³² is a protected amino group;and R¹⁴ is selected from the group consisting of —H and C₁-C₆ alkyl;wherein when R¹⁴ is C₁-C₆ alkyl, R¹⁴ is optionally substituted by one ormore moieties selected from the group consisting of cycloalkyl,heterocyclyl, aryl, and heteroaryl; wherein the treating is performedwith a deprotecting reagent, thereby producing the diamine compound.Protected amino groups useful in the present invention vary widely innature. Numerous protected amino groups useful in the present inventionfor either R³² or R³³ are described by Theodora W. Greene and Peter G.M. Wuts (Protective Groups in Organic Synthesis, 3rd ed., John Wiley &Sons, New York, 1999, pp. 494-653). For example either or both of R³²and R³³ can be a 4-chlorobenzylimino group. When R³³ is a4-chlorobenzylimino group, preferably R³² is —NH₂. In anotherembodiment, either or both of R³² and R³³ can be a t-butoxycarbonylaminogroup. When R³³ is a t-butoxycarbonylamino group, preferably R³² is—NH₂. In yet another embodiment, either or both of R³² and R³³ can be anN-phthalimido group. When R³³ is an N-phthalimido group, preferably R³²is —NH₂. In another embodiment, either or both of R³² and R³³ can be abenzyloxycarbonylamino group. In one preferred embodiment of the presentinvention, the protected amino group is any such group resulting fromthe reaction of an aldehyde with the corresponding amino group to form aSchiff base. A large variety of deprotecting reagents can beadvantageously used in the present invention to effect the conversion of24 to 22. Many such deprotecting reagents are described by Greene andWuts, supra. For example, when the protected amino group is a4-chlorobenzylimino group or a t-butoxycarbonylamino group, preferablythe deprotecting reagent is an acid. Some useful acid deprotectingagents include without limitation hydrochloric acid, hydrobromic acid,sulfuric acid, trifluoroacetic acid, phosphoric acid, phosphorus acid,and acetic acid. In another example, when R³² or R³³ is an N-phthalimidogroup, the deprotecting reagent can be either an acid or a base. Whenthe deprotecting reagent for the N-phthalimido group is a base, the basecan be, for example, a hydrazine, a metal sulfide, a metal hydroxide, ametal alkoxide, an amine, a hydroxylamine, and a metal amide complex.When the deprotecting reagent for the N-phthalimido group is an acid,the acid can be, for example, hydrochloric acid, hydrobromic acid,hydroiodic acid, sulfuric acid, methanesulfonic acid,trifluoromethanesulfonic acid, toluenesulfonic acid, benzenesulfonicacid, trifluoroacetic acid, phosphoric acid, phosphorus acid, or aceticacid. Preferably the treating of compound 24 with the deprotectingreagent is performed in the presence of water.

In the present reaction, R¹⁴ is preferably —H. R³³ preferably is —NH₂,or a salt thereof. R² is preferably methyl. In another preferredembodiment R¹, R⁵, R⁶, R⁷, R⁸, and R¹⁰ each is —H. A further preferredembodiment is one in which R³³ is a t-butoxycarbonylamino group. In aparticularly preferred embodiment, compound 24 has the structurecorresponding to Formula 25:

(or a salt thereof) wherein the parenthetical R means that the carbonalpha to the carboxylic acid function is in the Rabsolute configuration.In other words, compound 25 is the R-enantiomer or a salt thereof.

The present invention also provides a method for the preparation of theprotected diamine compound 24 (or a salt thereof) wherein the methodcomprises treating a sulfhydryl compound having the structure of Formula26:

with a protected amino ethyl alkylating compound having the structure ofFormula 27:

wherein R³⁴ is a nucleophilic substitution leaving group; therebyforming the protected diamine compound. Preferably, this reaction isperformed in the presence of a base. The base can be, for example, ahydrazine, a metal sulfide, a metal hydroxide, a metal alkoxide, anamine, a hydroxylamine, and a metal amide complex. Preferably the baseis an alkali metal hydroxide and more preferably the base is potassiumhydroxide or sodium hydroxide. R³⁴ in the structure of Formula 27 canvary widely and can represent essentially any nucleophilic leaving groupwhich produces either a pharmaceutically acceptable anion or an anionwhich can be exchanged for a pharmaceutically acceptable anion. In otherwords, (R³⁴)⁻ is a pharmaceutically acceptable anion or an anion whichcan be exchanged for a pharmaceutically acceptable anion. For example,R³⁴ can be chloro, bromo, iodo, methanesulfonato, toluenesulfonato,benzenesulfonato, or trifluoromethanesulfonato. Preferably R³⁴ ischloro, bromo, or iodo and more preferably R³⁴ is bromo. In the presentreaction it is preferred for R³³ to be —NH₂. In the present reaction R²can be C₁-C₆ alkyl optionally substituted by one or more substituentselected from the group consisting of —OH, alkoxy and halogen.Preferably R² is C₁-C₃ alkyl optionally substituted by one or moresubstituent selected from the group consisting of —OH, alkoxy andhalogen, and more preferably R² is C₁-C₃ alkyl. For example, R²advantageously can be methyl. In a further embodiment R⁵ and R⁶ each canbe —H. In yet another embodiment R¹ and R⁷ independently can be selectedfrom the group consisting of H and C₁-C₆ alkyl optionally substituted byone or more halogen; preferably R¹ and R⁷ each is —H. In the presentreaction, R³² preferably can be selected from the group consisting of a4-chlorobenzylimino group, a t-butoxycarbonylamino group, and anN-phthalimido group, and more preferably R³² is a t-butoxycarbonylaminogroup.

The present invention also provides a method for the preparation of asulflhydryl compound having the structure of Formula 28:

wherein R², R⁵, and R⁶ are as defined above, and wherein the methodcomprises treating under hydrolysis conditions a thiazolidine compoundhaving the structure of Formula 29:

or a salt thereof, wherein R³⁵ is a moiety selected from the groupconsisting of —H and C₁-C₆ alkyl; thereby forming the sulthydrylcompound. The hydrolysis conditions preferably comprise contacting thethiazolidine compound with an acid in the presence of water. Preferablythe acid is selected from the group consisting of hydrochloric acid,hydrobromic acid, hydroiodic acid, sulfuric acid, methanesulfonic acid,trifluoromethanesulfonic acid, toluenesulfonic acid, benzenesulfonicacid, trifluoroacetic acid, phosphoric acid, phosphorus acid, and aceticacid. In one embodiment of the present reaction R⁵ and R⁶ each is —H. Inanother embodiment R² of compound 29 is C₁-C₆ alkyl optionallysubstituted by one or more substituent selected from the groupconsisting of —OH, alkoxy, and halogen. Preferably R² is C₁-C₃ alkyloptionally substituted by one or more substituent selected from thegroup consisting of alkoxy and halogen, and more preferably R² is C₁-C₃alkyl. For example, R² can be methyl. In another embodiment of thepresent invention R³⁵ of compound 29 is methyl.

The present invention also provides a method for the preparation of amethyl thiazolidine compound having the structure of Formula 30:

(or a pharmaceutically-acceptable salt thereof) wherein R³⁶ is C₁-C₆alkyl; wherein the method comprises treating under methylationconditions a deprotonatable thiazolidine compound having the structureof Formula 31:

thereby forming the methyl thiazolidine compound. Preferably themethylation conditions comprise treating the deprotonatable thiazolidinecompound with a base and a methylating agent. The nature of the base canvary widely. The base can be, for example, a metal hydroxide, a metalalkoxide, a metal hydride, a metal alkyl, and a metal amide complex.Preferably the base is a metal amide complex. Some metal amide complexesuseful as a base in the present invention include without limitationlithium hexamethyldisilazide, sodium hexamethyldisilazide, potassiumhexamethyldisilazide, lithium diisopropyl amide, sodium diisopropylamide, potassium diisopropyl amide, sodium amide, lithium amide,potassium amide, sodium diethylamide, lithium diethylamide, potassiumdiethylamide, methyl lithium, t-butyl lithium, sec-butyl lithium, methylsodium, t-butyl sodium, sec-butyl sodium, and methyl magnesium bromide.In the present methylation reaction R³⁶ can be C₁-C₃ alkyl; for exampleR³⁶ can be methyl.

The present invention further provides a method for the preparation ofthe deprotonatable thiazolidine compound 31 wherein the method comprisescontacting under condensing conditions a cysteine C₁-C₆ alkyl ester withpivalaldehyde, thereby forming the deprotonatable thiazolidine compound.Preferably condensing conditions comprise performing the contacting inthe presence of a base. The base can vary widely. For example the basecan be, without limitation, a hydrazine, a metal sulfide, a metalhydroxide, a metal alkyl base, a metal alkoxide, an amine, ahydroxylamine, and a metal amide complex. When the base is a metal amidecomplex, it can be, for example, lithium bis(trimethylsilyl)amide. Inthe present condensation reaction it is preferred that R³⁶ is C₁-C₃alkyl, and more preferably R³⁶ is methyl.

In another embodiment the present invention provides a method for thepreparation of an alpha-amino acid compound having the structure ofFormula 32:

(or a salt, an enantiomer, or a racemate thereof) wherein: R² isselected from the group consisting of C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆alkynyl, C₁-C₅ alkoxy-C₁ alkyl, and C₁-C₅ alkylthio-C₁ alkyl; R¹, R⁵,R⁶, and R⁷ independently are selected from the group consisting of —H,halogen, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, and C₁-C₅ alkoxy-C₁alkyl; R⁹ and R¹⁰ independently are selected from the group consistingof —H, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, and C₁-C₅ alkoxy-C₁alkyl; and when any of R¹, R², R⁵, R⁶, R⁷, R⁹, and R¹⁰ independently isa moiety selected from the group consisting of alkyl, alkenyl, andalkynyl, then the moiety is optionally substituted by one or moresubstituent selected from the group consisting of —OH, alkoxy, andhalogen; wherein the method comprises treating under hydrolyzingconditions a hydantoin compound having the structure of Formula 33:

(or a salt, an enantiomer, or a racemate thereof), thereby forming thealpha-amino acid compound. The hydrolyzing conditions can comprise, forexample, contacting the hydantoin compound with an acid to produce anacid hydrolyzate. Acids useful in the present hydrolysis reactioninclude, for example, hydrochloric acid, hydrobromic acid, hydroiodicacid, sulfuric acid, trifluoroacetic acid, or phosphoric acid. Themethod for the preparation of alpha-amino acid compound 32 can furthercomprise treating the acid hydrolyzate with an ion exchange resin.Alternatively, the hydrolyzing conditions can comprise contacting thehydantoin compound with a base to produce a base hydrolyzate. Basesuseful in the present base hydrolysis include without limitation ahydrazine, a metal sulfide, a metal hydroxide, or a metal alkoxide.Whether the hydrolysis is base-mediated or acid-mediated, it ispreferred that R¹, R⁵, R⁶, and R⁷ each is —H in the structure ofcompound 33. It is also preferred that R⁹ and R¹⁰ each is —H. In aparticularly preferred embodiment, the alpha-amino acid compound has thestructure of Formula 34:

(or a salt, an enantiomer, or a racemate thereof).

The present invention further provides a method for the preparation of ahydantoin compound having the structure of Formula 35:

(or a salt, an enantiomer, or a racemate thereof) wherein R¹, R², R⁵,R⁶, R⁷, R⁹, and R¹⁰ are as defined above, wherein the method comprisescontacting an alpha-sulfo ketone compound having the structure ofFormula 36:

with a source of cyanide in the presence of a source of ammoniumcarbonate and water, thereby producing the hydantoin compound. Thesource of cyanide can be, for example, hydrogen cyanide or a metalcyanide salt. When the source of cyanide is a metal cyanide salt,preferably the salt is sodium cyanide, potassium cyanide, or lithiumcyanide. More preferably the metal cyanide salt is sodium cyanide. Forcompound 36 in the present hydantoin-forming reaction, R¹¹, R⁵, R⁶, andR⁷ each is preferably —H. It is further preferred that R⁹ and R¹⁰ eachis —H. The method of preparing compound 35 can further comprise a chiralseparation step. When the method of preparing compound 35 furthercomprises a chiral separation step, then the hydantoin compound productpreferably has the structure of compound 33 (or a salt or an enantiomerthereof).

Also provided by the present invention is a method for the preparationof an alpha-sulfo ketone compound 36 wherein the method comprisescontacting an aminothiol compound having the structure of Formula 37:

with di-t-butyl carbonate in the presence of a base to produce anintermediate mixture; and contacting the intermediate mixture with analpha-chloro ketone compound having the structure of Formula 38:

thereby producing the alpha-sulfo ketone compound. In the presentreaction the base can vary widely. For example, the base can be withoutlimitation a hydrazine, a metal sulfide, a metal hydroxide, a metalalkoxide, an amine, a hydroxylamine, and a metal amide complex.Preferably the base is a metal hydroxide such as sodium hydroxide,potassium hydroxide, or lithium hydroxide. In the present reaction it ispreferred that R¹, R⁵, R⁶, and R⁷ each is —H. It is further preferredthat R⁹ and R¹⁰ each is —H.

The term “pharmaceutically-acceptable salts” embraces salts commonlyused to form alkali metal salts and to form addition salts of free acidsor free bases. The nature of the salt is not critical, provided that itis pharmaceutically acceptable. Pharmaceutically acceptable salts areparticularly useful as products of the methods of the present inventionbecause of their greater aqueous solubility relative to a correspondingparent or neutral compound. Such salts must have a pharmaceuticallyacceptable anion or cation. Suitable pharmaceutically-acceptable acidaddition salts of compounds of the present invention may be preparedfrom inorganic acid or from an organic acid. Examples of such inorganicacids are hydrochloric, hydrobromic, hydroiodic, nitric, carbonic,sulfuric and phosphoric acid. Appropriate organic acids include fromaliphatic, cycloaliphatic, aromatic, araliphatic, heterocyclic,carboxylic and sulfonic classes of organic acids, examples of which areformic, acetic, propionic, succinic, glycolic, gluconic, lactic, malic,tartaric, citric, ascorbic, glucoronic, maleic, fumaric, pyruvic,aspartic, glutamic, benzoic, anthranilic, mesylic, salicylic,p-hydroxybenzoic, phenylacetic, mandelic, embonic (pamoic),methanesulfonic, ethylsulfonic, benzenesulfonic, sulfanilic, stearic,cyclohexylaminosulfonic, algenic, galacturonic acid. Suitablepharmaceutically-acceptable base addition salts of compounds of thepresent invention include metallic salts made from aluminum, calcium,lithium, magnesium, potassium, sodium and zinc or organic salts madefrom N,N′-dibenzylethyleneldiamine, choline, chloroprocaine,diethanolamine, ethylenediamine, meglumine (N-methylglucamine) andprocain. Suitable pharmaceutically acceptable acid addition salts of thecompounds of the present invention when possible include those derivedfrom inorganic acids, such as hydrochloric, hydrobromic, boric,fluoroboric, phosphoric, metaphosphoric, nitric, carbonic (includingcarbonate and hydrogen carbonate anions), sulfonic, and sulfuric acids,and organic acids such as acetic, benzenesulfonic, benzoic, citric,ethanesulfonic, fumaric, gluconic, glycolic, isothionic, lactic,lactobionic, maleic, malic, methanesulfonic, trifluoromethanesulfonic,succinic, toluenesulfonic, tartaric, and trifluoroacetic acids. Suitablepharmaceutically acceptable base salts include ammonium salts, alkalimetal salts such as sodium and potassium salts, and alkaline earth saltssuch as magnesium and calcium salts. All of these salts may be preparedby conventional means from the corresponding conjugate base or conjugateacid of the compounds of the present invention by reacting,respectively, the appropriate acid or base with the conjugate base orconjugate acid of the compound. Another pharmaceutically acceptable saltis a resin-bound salt.

While it may be possible for the compounds of the present invention tobe administered as the raw chemical, it is preferable to present them asa pharmaceutical composition. According to a further aspect, the presentinvention provides a pharmaceutical composition comprising a compound ofthe present invention or a pharmaceutically acceptable salt or solvatethereof, together with one or more pharmaceutically acceptable carriersthereof and optionally one or more other therapeutic ingredients. Thecarrier(s) must be acceptable in the sense of being compatible with theother ingredients of the formulation and not deleterious to therecipient thereof.

The formulations include those suitable for oral, parenteral (includingsubcutaneous, intradermal, intramuscular, intravenous andintraarticular), rectal and topical (including dermal, buccal,sublingual and intraocular) administration although the most suitableroute may depend upon for example the condition and disorder of therecipient. The formulations may conveniently be presented in unit dosageform and may be prepared by any of the methods well known in the art ofpharmacy. All methods include the step of bringing into association acompound of The present invention or a pharmaceutically acceptable saltor solvate thereof with the carrier which constitutes one or moreaccessory ingredients. In general, the formulations are prepared byuniformly and intimately bringing into association the active ingredientwith liquid carriers or finely divided solid carriers or both and then,if necessary, shaping the product into the desired formulation.

Formulations of the present invention suitable for oral administrationmay be presented as discrete units such as capsules, cachets or tabletseach containing a predetermined amount of the active ingredient; as apowder or granules; as a solution or a suspension in an aqueous liquidor a non-aqueous liquid; or as an oil-in-water liquid emulsion or awater-in-oil liquid emulsion. The active ingredient may also bepresented as a bolus, electuary or paste.

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared bycompressing in a suitable machine the active ingredient in afree-flowing form such as a powder or granules, optionally mixed with abinder, lubricant, inert diluent, lubricating, surface active ordispersing agent. Molded tablets may be made by molding in a suitablemachine a mixture of the powdered compound moistened with an inertliquid diluent. The tablets may optionally be coated or scored and maybe formulated so as to provide slow or controlled release of the activeingredient therein.

Formulations for parenteral administration include aqueous andnon-aqueous sterile injection solutions which may contain anti-oxidants,buffers, bacteriostats and solutes which render the formulation isotonicwith the blood of the intended recipient; and aqueous and non-aqueoussterile suspensions which may include suspending agents and thickeningagents. The formulations may be presented in unit-dose or multi-dosecontainers, for example sealed ampoules and vials, and may be stored ina freeze-dried (lyophilized) condition requiring only the addition ofthe sterile liquid carrier, for example, saline, water-for-injection,immediately prior to use. Extemporaneous injection solutions andsuspensions may be prepared from sterile powders, granules and tabletsof the kind previously described.

Formulations for rectal administration may be presented as a suppositorywith the usual carriers such as cocoa butter or polyethylene glycol.

Formulations for topical administration in the mouth, for examplebuccally or sublingually, include lozenges comprising the activeingredient in a flavored basis such as sucrose and acacia or tragacanth,and pastilles comprising the active ingredient in a basis such asgelatin and glycerin or sucrose and acacia.

Preferred unit dosage formulations are those containing an effectivedose, as hereinbelow recited, or an appropriate fraction thereof, of theactive ingredient.

It should be understood that in addition to the ingredients particularlymentioned above, the formulations of this invention may include otheragents conventional in the art having regard to the type of formulationin question, for example those suitable for oral administration mayinclude flavoring agents.

The compounds of the invention may be administered orally or viainjection at a dose of from 0.001 to 2500 mg/kg per day. The dose rangefor adult humans is generally from 0.005 mg to 10 g/day. Tablets orother forms of presentation provided in discrete units may convenientlycontain an amount of compound of the invention which is effective atsuch dosage or as a multiple of the same, for instance, units containing5 mg to 500 mg, usually around 10 mg to 200 mg.

The compounds of Formula 1 are preferably administered orally or byinjection (intravenous or subcutaneous). The precise amount of compoundadministered to a patient will be the responsibility of the attendantphysician. However, the dose employed will depend on a number offactors, including the age and sex of the patient, the precise disorderbeing treated, and its severity. Also, the route of administration mayvary depending on the condition and its severity.

Compounds of the present invention can exist in tautomeric, geometric orstereoisomeric forms. The present invention contemplates all suchcompounds, including cis- and trans-geometric isomers, E- andZ-geometric isomers, R- and S-enantiomers, diastereomers, d-isomers,l-isomers, the racemic mixtures thereof and other mixtures thereof, asfalling within the scope of the invention. Pharmaceutically acceptablesales of such tautomeric, geometric or stereoisomeric forms are alsoincluded within the invention.

The terms “cis” and “trans” denote a form of geometric isomerism inwhich two carbon atoms connected by a double bond will each have twohighest ranking groups on the same side of the double bond (“cis”) or onopposite sides of the double bond (“trans”). Some of the compoundsdescribed contain alkenyl groups, and are meant to include both cis andtrans or “E” and “Z” geometric forms.

Some of the compounds described contain one or more stereocenters andare meant to include R, S, and mixtures of R and S forms for eachstereocenter present.

The following general synthetic sequences are useful in making thepresent invention.

The following structures are illustrative of the many compounds providedby the present invention. The examples provided here are not intended tobe limiting and one of skill in the art, given the present disclosure,will recognize that many alternative structures are embraced by thepresent invention.

The following compounds are further examples of compounds embraced bythe present invention or useful in the preparation of compounds of thepresent invention.

(2R,4R)-Methyl-2-tert-butyl-1,3-thiazoline-3-formyl-4-carboxylate.

(2R,4R)-Methyl-2-tert-butyl-1,3-thiazoline-3-formyl-4-methyl-4-carboxylate.

S-[2-[[(1,1-Dimethylethoxy)carbonyl]amino]ethyl]-2-methyl-L-cysteinetrifluoroacetate.

(S)-1-[(Benzyloxycarbonyl)amino]-2-propanol.

(S)-1-[(Benzyloxycarbonyl)amino]-2-propanol tosylate.

S-[(1R)-2-(Benzyloxycarbonylamino)-1-methylethyl]-2-methyl-L-cysteinetrifluoroacetate.

S-[(1R)-2-Amino-1-methylethyl]-2-methyl-L-cysteine hydrochloride.

S-(2-Aminoethyl)-L-cysteine, methyl ester.

N-{4-Chlorophenyl)methylene]-S-[2-[[(4-chlorophenyl)methylene]amino]ethyl]-L-cysteine,methyl ester.

N-[4-Chlorophenyl)methylene]-S-[2-[[(4-chlorophenyl)methylene]amino]ethyl]-2-methyl-D/L-cysteine,methyl ester.

The following examples are provided to illustrate the present inventionand are not intended to limit the scope thereof. Those skilled in theart will readily understand that known variations of the conditions andprocesses of the following preparative procedures can be used to preparethese compounds.

EXAMPLE 1

S-[2-[(1-Iminoethyl)amino]ethyl]-2-methyl-L-cysteine, dihydrochlorideExample-1A(2R,4R)-Methyl-2-tert-butyl-1,3-thiazoline-3-formyl-4-carboxylate

See Jeanguenat and Seebach, J. Chem. Soc. Perkin Trans. 1, 2291 (1991)and Pattenden et al. Tetrahedron, 49, 2131 (1993): (R)-cysteine methylester hydrochloride (8.58 g, 50 mmol), pivalaldehyde (8.61 g, 100 mmol),and triethylamine (5.57 g, 55 mmol) were refluxed in pentane (800 ml)with continuous removal of water using a Dean-Stark trap. The mixturewas filtered and evaporated. The resultant thiazolidine (9.15 g, 45mmol) and sodium formate (3.37 g, 49.5 mmol) were stirred in formic acid(68 ml) and treated with acetic anhydride (13 mL, 138 mmol), dropwiseover 1 hour at 0-5° C. The solution was allowed to warm to RT and stirovernight. The solvents were evaporated and the residue was neutralizedwith aqueous 5% NaHCO₃ and extracted with ether (3×). The combinedorganic layers were dried (anhy. MgSO₄), filtered, and evaporated togive the title compound which was crystallized from hexane-ether aswhite crystals (8.65 g) (80% overall, 8:1 mixture of conformers). ¹H NMR(CDCl₃) δ major conformer: 1.04 (s, 9H), 3.29 (d, 1H), 3.31 (d, 1H),3.78 (s, 3H), 4.75 (s, 1H), 4.90 (t, 1H), 8.36 (s, 1H). MS m/z(electrospray) 232 (M+H)⁺ (100%), 204 (10) 164 (24).

Example-1B(2R,4R)-Methyl-2-tert-butyl-1,3-thiazoline-3-formyl-4-methyl-4-carboxylate

To a solution of the product of Example-1A,(2R,4R)-Methyl-2-tert-butyl-1,3-thiazoline-3-formyl-4-carboxylate (8.65g, 37.4 mmol), in anhydrous tetrahydrofuran (130 mL) under N₂ at −78° C.was added DMPU (25 mL) and the mixture stirred for 5 min. Lithiumbis(trimethylsilyl)amide, 1 M in tetrahydrofuran, (37.5 mL), was added,and the mixture stirred for 30 min. After methyl iodide (5.84 g, 41.1mmol) was added, the mixture was held at −78° C. for 4 hr and thenwarmed to room temperature with continuous stirring. The solvents wereevaporated in vacuo and brine and ethyl acetate was added. The aqueousphase was extracted 3×EtOAc, and the combined organic layers were washedwith 10% KHSO₄, water, and brine. They were then dried (anhy. MgSO₄),filtered, and stripped of all solvent under reduced pressure.Chromatography of the residual oil on silica with 1-10% EtOAc/hexaneyielded the title compound (5.78 g, 63%, 2.4:1 mixture of conformers).¹H NMR (CDCl₃) δ major conformer, 1.08 (s, 9H), 1.77 (s, 3H), 2.72 (d,1H), 3.31 (d, 1H), 377 (s, 3H), 4.63 (s, 1H), 8.27 (s, 1H); minorconformer, 0.97 (s, 9H), 1.79 (s, 3H), 2.84 (d, 1H), 3.63 (d, 1H), 3.81(s, 3H), 5.29 (s, 1H), 8.40 (s, 1H); MS m/z (electrospray) 246 (M+H)⁺(100%), 188 (55) 160 (95). Retention time of 16.5 min on a DaicelChemical Industries Chiracel OAS column, 10-40% IPA/hexane 0-25min, >95% ee.

Example-1C (2R) 2-Methyl-L-cysteine hydrochloride

The product of Example-1B,(2R,4R)-Methyl-2-tert-butyl-1,3-thiazoline-3-formyl-4-methyl-4-carboxylate,(5.7 g, 23.2 mmol) was stirred with 6N HCl (100 mL) under N₂ and held atvigorous reflux for 2 days. The solution was cooled, washed with EtOAcand evaporated to yield the product (2R) 2-methyl-cysteine hydrochloride(3.79 g, 95%) as a light yellow powder. ¹H NMR (DMSO-d₆) δ 1.48 (s, 3H)2.82 (t, 1H), 2.96 (bs, 2H), 8.48 (s, 3H). MS m/z (electrospray) 136[M+H⁺].

Example-1DS-[2-[[(1,1-dimethylethoxy)carbonyl]amino]ethyl]-2-methyl-L-cysteinetrifluoroacetate

Sodium hydride (2.6 g, 60% in mineral oil, 65 mmol) was added to anoven-dried, vacuum-cooled RB flask, containing oxygen-free1-methyl-2-pyrrolidinone (5 mL). The mixture was cooled to −10° C. andstirred under N₂. The product of Example-1C, 2-Methyl-L-cysteinehydrochloride, (3.6 g, 21.0 mmol) dissolved in oxygen-free1-methyl-2-pyrrolidinone (25 ml), was added in portions. After all H₂evolution ceased, 2-[(1,1-dimethylethoxycarbonyl)-amino]ethyl bromide(4.94 g, 21 mmol) in oxygen-free 1-methyl-2-pyrrolidinone (15 mL) wasadded at −10° C. The reaction was then stirred for 4 hr allowing warmingto room temperature. The solution was neutralized with 1 N HCl and the1-methyl-2-pyrrolidinone was removed by evaporation in vacuo.Reverse-phase chromatography with 1-20% acetonitrile in 0.05% aqueoustrifluoro acetic acid solution yielded the title compound (5.9 g),recovered by freeze-drying appropriate fractions. ¹H NMR (DMSO-d₆/D₂O) δ1.31 (s, 9H), 1.39 (s, 3H), 2.55 (m, 2H), 2.78 (d, 1H), 3.04 (d, 1H),3.06 (t, 2H). HRMS calc. for C₁₁H₂₂N₂O₄S: 279.1375 (M+H⁺), found279.1379.

Example-1E S-(2-aminoethyl)-2-methyl-L-cysteine hydrochloride

The product of Example-1D,S-[2-[[(1,1-dimethylethoxy)carbonyl]amino]ethyl]-2-methyl-L-cysteinetrifluoroacetate, (5.5 g, 14.0 mmol) was dissolved in 1 N HCl (100 mL)and stirred at room temperature under nitrogen overnight. The solutionwas removed by freeze-drying to give the titleS-(2-aminoethyl)-2-methyl-L-cysteine hydrochloride, ¹H NMR (DMSO-d₆/D₂O)δ 1.43 (s, 3H), 2.72 (m, 2H), 2.85 (d, 1H), 2.95 (t, 2H), 3.07 (d, 1H).m/z [M+H⁺] 179.

The product of Example-1E, was dissolved in H₂O, the pH adjusted to 10with 1 N NaOH, and ethyl acetimidate hydrochloride (1.73 g, 14.0 mmol)was added. The reaction was stirred 15-30 min, the pH was raised to 10,and this process repeated 3 times. The pH was adjusted to 3 with HCl andthe solution loaded onto a washed DOWEX 50WX4-200 column. The column waswashed with H₂O and 0.25 M NH₄OH, followed by 0.5 M NH₄OH. Fractionsfrom the 0.5 M NH₄OH wash were immediately frozen, combined andfreeze-dried to give an oil that was dissolved in 1N HCl and evaporatedto give the title compound as a white solid (2.7 g). ¹H NMR(DMSO-d₆/D₂O) δ 1.17 (s, 3H), 2.08 (s, 3H), 2.52 (d, 1H), 2.68 (m, 2H),2.94 (d, 1H), 3.23 (t, 2H). HRMS calc. for C₈H₁₈N₃O₂S: 220.1120 [M+H⁺],found 220.1133.

EXAMPLE 2

2-[[[2-[(1-Iminoethyl)amino]ethyl]thio]methyl]-O-methyl-D-serine,dihydrochloride

The procedures and methods utilized in this example were identical tothose of Example 1 except that in step Example-1B methoxymethyl iodidewas used instead of methyl iodide. These procedures yielded the titleproduct as a white solid (2.7 g). ¹H NMR (D₂O) δ 2.06 (s, 3H), 2.70 (m,3H), 3.05 (d, 1H), 3.23 (s, 3H), 3.32 (t, 2H), 3.46 (d, 1H), 3.62 (d,1H). HRMS calc. for C₉H₂₀N₃O₃S: 250.1225 [M+H⁺], found 250.1228.

EXAMPLE 3

S-[(1R)-2-[(1-Iminoethyl)amino]-1-methylethyl]-2-methyl-L-cysteine,dihydrochloride Example-3A (S)-1-[(benzyloxycarbonyl)amino]-2-propanol

To a solution of (S)-1-amino-2-propanol (9.76 g, 130 mmol) in anhydrousbenzene (60 mL) at 0° C. was added benzyl chloroformate (10.23 g, 60mmol) in anhydrous benzene (120 mL) slowly, in portions, over a periodof 20 min while vigorously stirring under an atmosphere of nitrogen. Themixture was stirred for 1 hour at 0° C., then allowed to warm to roomtemperature and stirred for a further 2 hours. The mixture was washedwith water (2×) and brine (2×) before the organic layer was dried overanhydrous MgSO₄. Evaporation of all solvent gave the title product as anoil. ¹H NMR (CDCl₃) δ 1.22 (d, 3H,) 2.40 (bs, 1H), 3.07 (m, 1H), 3.37(m, 1H)), 3.94 (m, 1H), 5.16 (s, 2H), 5.27 (m, 1H), 7.38 (m, 5H). MS m/z(electrospray) 232 [M+23]⁺ (100%), 166 (96).

Example-3B (S)-1-[(benzyloxycarbonyl)amino]-2-propanol tosylate

To a solution of the product of Example-3A,(S)-1-[(benzyloxycarbonyl)amino]-2-propanol, (9.74 g, 46.7 mmol) andtriethylamine 7.27 g, 72 mmol) in methylene chloride (60 mL) at 0° C.was added toluene sulfonyl chloride (9.15 g, 48 mmol) in methylenechloride (18 mL) slowly, in portions, over a period of 20 min whilevigorously stirring under nitrogen. The mixture allowed to warm to roomtemperature and stirred for a further 36 hours under nitrogen. Theorganic layer was washed with 1N HCl, water, 5% NaHCO₃ solution, waterand brine before it was dried over anhydrous MgSO₄. Evaporation of allsolvent gave a white solid which was passed though a silica plug withethyl acetate/hexane (1:4) to remove excess toluene sulfonyl chlorideand then with ethyl acetate/hexane (1:3) to give the title product aswhite crystals. This material was recrystallized from ethylacetate/hexane to give white needles (10.8 g). ¹H NMR (CDCl₃) δ 1.22 (d,3H,) 2.39 (s, 3H), 3.20 (m, 1H), 3.43 (dd, 1H)), 4.66 (m, 1H), 5.02 (m,1H), 5.04 (ABq, 2H), 7.34 (m, 7H), 7.77 (d, 2H). MS m/z (electrospray)386 [M+23]⁺ (100%), 320 (66). The product was examined on a RegisTechnologies Inc. Perkle Covalent (R,R) β-GEM1 HPLC column using mobilephase of isopropanol/hexane and a gradient of 10% isopropanol for 5 min,then 10 to 40% isopropanol over a period of 25 min, and using both UVand Laser Polarimetry detectors. Retention time major peak: 22.2min, >98% ee.

Example-3CS-[(1R)-2-(Benzyloxycarbonylamino)-1-methylethyl]-2-methyl-L-cysteinetrifluoroacetate

The product of Example-1C, 2-methyl-L-cysteine hydrochloride, (1 g, 6.5mmol) was added to an oven dried, N₂ flushed RB flask, dissolved inoxygen-free 1-methyl-2-pyrrolidinone (5 mL), and the system was cooledto 0° C. Sodium hydride (0.86 g, 60% in mineral oil, 20.1 mmol) wasadded and the mixture was stirred at 0° C. for 15 min. A solution of theproduct of Example-3B, (2S)-1-[(N-benzyloxycarbonyl)amino]-2-propanoltosylate (2.5 g, 7 mmol) dissolved in oxygen-free1-methyl-2-pyrrolidinone (10 mL) was added over 10 min. After 15 min at0° C., the reaction mixture was stirred at room temperature for 4.5hours. The solution was then acidified to pH 4 with 1N HCl and1-methyl-2-pyrrolidinone was removed by evaporation in vacuo. Reversephase chromatography with 20-40% acetonitrile in 0.05% aqueous trifluoroacetic acid solution yielded the title compound in (0.57 g), recoveredby freeze-drying. ¹H NMR (H₂O, 400 MHz) δ 1.0 (d, 3H), 1.4 (s, 3H), 2.6(m, 2H), 2.8 (m, 1H), 3.1 (m, 2H), 3.6 (s, 1H), 5.0 (ABq, 2H), 7.3 (m,5H). MS m/z (electrospray): 327 [M+H⁺] (100%), 238 (20), 224 (10), and100 (25).

Example-3D S-[(1R)-2-Amino-1-methylethyl]-2-methyl-L-cysteinehydrochloride

The product of Example-3C,S-[(1R)-2-(Benzyloxycarbonylamino)-1-methylethyl]-2-methyl-L-cysteinetrifluoroacetate, (0.5 g, 1.14 mmol) was dissolved in 6N HCl andrefluxed for 1.5 hour. The mixture was then cooled to room temperatureand extracted with EtOAc. The aqueous layer was concentrated in vacuo togive the title product, (2R, 5R)-S-(1-amino-2-propyl)-2-methyl-cysteinehydrochloride (0.29 g), which was used without further purification. ¹HNMR (H₂O, 400 MHz) δ 1.2 (m, 3H), 1.4 (m, 3H), 2.7 (m, 1H), 2.8-3.2 (m,2H), 3.4 (m, 1H). (some doubling of peaks due to rotameric forms). MSm/z (electrospray): 193 [M+H⁺] (61%), 176 (53), 142 (34), 134 (100), and102 (10).

The product of Example-3D,S-[(1R)-2-Amino-1-methylethyl]-2-methyl-L-cysteine hydrochloride, (0.2g, 0.76 mmol) was dissolved in 2 mL of H₂O, the pH was adjusted to 10.0with 1N NaOH, and ethyl acetimidate hydrochloride (0.38 g, 3 mmol) wasadded in four portions over 10 minutes, adjusting the pH to 10.0 with 1NNaOH as necessary. After 1 h, the pH was adjusted to 3 with 1N HCl. Thesolution was loaded onto a water-washed DOWEX 50WX4-200 column. Thecolumn was washed with H₂O and 0.5N NH₄OH. The basic fractions werepooled and concentrated to dryness in vacuo. The residue was acidifiedwith 1N HCl and concentrated to the Example 3 title product, (49 mg). ¹HNMR (H₂O, 400 MHz) δ 1.3-1.0 (m, 3H), 1.5 (m, 3H), 2.1-1.8 (m, 3H),3.4-2.6 (m, 5H), 3.6 (m, 1H) (rotamers observed). MS m/z (electrospray):234 [M+H⁺] (100%), 176 (10), and 134 (10).

EXAMPLE 4

S-[(1S)-2-[(1-Iminoethyl)amino]-1-methylethyl]-2-methyl-L-cysteine,dihydrochloride

The procedures and methods employed here were identical to those ofExample 3, except that in step Example-3A (R)-1-amino-2-propanol wasused instead of (S)-1-amino-2-propanol to give the title material,S-[(1S)-2-[(1-Iminoethyl)amino]-1-methylethyl]-2-methyl-L-cysteinehydrochloride. ¹H NMR (H₂O,400 MHz) δ 3.6 (m, 1H), 3.4-2.6 (m, 5H),2.1-1.8 (m, 3H), 1.5 (m, 3H), and 1.3-1.0 (m, 3H). HRMS calc forC₉H₁₉N₃O₂S [M+H⁺]: 234.1276. Found: 234.1286.

EXAMPLE 5

S-[(1R/S)-2-[(1-Iminoethyl)amino]-1-ethylethyl]-2-methyl-L-cysteine,dihydrochloride

The procedures and methods utilized in this synthesis are identical tothose of Example 3, except that in step Example-3A(R/S)-1-amino-2-butanol is used instead of (S)-1-amino-2-propanol.

EXAMPLE 6

S-[(1S)-2-[(1-iminoethyl)amino]-1-(fluoromethyl)ethyl]-2-methyl-L-cysteine,dihydrochloride

A sample of 2-[(2R)-Oxiranylmethyl]-1H-isoindole-1,3-dione (G. Alexanderet al. Tetrahedron Asymmetry, 7, 1641-8, 1996) is treated with potassiumhydrogen difluoride to give2-[(2R)-3-fluoro-2-hydroxypropyl]-1H-isoindole-1,3-dione in the presenceof catalyst nBu₄NH₂F₃. The procedures and methods used in this synthesisare identical to those of Example 3, except that in step Example-3B2-[(2R)-3-fluoro-2-hydroxypropyl]-1H-isoindole-1,3-dione is used insteadof (S)-1-[(benzyloxycarbonyl)amino]-2-propanol to produce the titleproduct.

EXAMPLE 7

S-[2-[(1-Iminoethyl)amino]ethyl]-2-ethyl-L-cysteine, dihydrochloride

The procedures and methods used in this synthesis were the same as thoseused in Example 1 except that ethyl triflate was used in Example-1Binstead of methyl iodide. Reverse phase chromatography, using a gradientof 10-40% acetonitrile in water, was used to purify the title product(20% yield). ¹H NMR (D₂O) δ 0.83 (t, 3H), 1.80 (m, 2H), 2.08 (s, 3H),2.68 (m, 1H), 2.78 (m, 1H), 2.83 (m, 1H), 3.11 (m, 1H), 3.36 (t, 2H).HRMS calc. for C₉H₂₀N₃O₂S: 234.1276 [M+H⁺], found 234.1284.

EXAMPLE 8

S-[(1R)-2-[(1-Iminoethyl)amino]-1-methylethyl]-2-ethyl-L-cysteine,dihydrochloride

The procedures and methods employed in this synthesis are the same asthose used in Example 1 except that ethyl triflate is used in Example-1Binstead of methyl iodide. The 2-ethyl-L-cysteine hydrochloride thusprepared is treated as described in the procedures and methods ofExamples 3C-3E to give the title compound.

EXAMPLE 9

S-[(1R/S)-2-[(1-Iminoethyl)amino]-1-ethylethyl]-2-ethyl-L-cysteine,dihydrochloride

The procedures and methods utilized in this synthesis are the same asthose used in Example 5 except that 2-ethyl-L-cysteine hydrochloride(prepared in Example 7) is used instead of 2-methyl-L-cysteinehydrochloride to give the title compound.

EXAMPLE 10

S-[(1S)-2-[(1-iminoethyl)amino]-1-fluoromethylethyl]-2-ethyl-L-cysteine,dihydrochloride

The procedures and methods utilized in this synthesis are the same asthose used in Example 6, except that (2R)-2-ethyl cysteine hydrochloride(prepared in Example 7) is used instead of (2R)-2-methyl cysteinehydrochloride, to give the title compound.

EXAMPLE 11

2-[[[[2-(1-Iminoethyl)amino]ethyl]thio]methyl]-D-valine, dihydrochlorideExample-11a Isopropyl Triflate

Silver triflate (25.25 g, 98.3 mmol) stirred in diethyl ether (300 mL)under nitrogen was treated with isopropyl iodide (16.54 g, 98.5 mmol) inether (200 mL) over 15 minutes. The mixture was stirred for 10 minutesand then filtered. The filtrate was distilled at reduced pressure. Thedistillate was redistilled at atmospheric pressure to remove themajority of the diethyl ether, leaving a mixture of the title isopropyltriflate-diethyl ether (84:16 by weight) (15.64 g, 70% corrected) as acolorless liquid. ¹H NMR (CDCl₃, 400 MHz) δ 1.52 (d, 6H), 5.21 (septet,1H).

The procedures and methods utilized here were the same as those used inExample 1 except that isopropyl triflate replaced methyl iodide inExample-1B. The crude title product was purified by reversed phasechromatography using a gradient elution of 10-40% acetonitrile in water.¹H NMR (H₂O, 400 MHz) δ 0.94 (dd, 6H), 2.04 (septet, 1H), 2.10 (s, 3H),2.65, 2.80 (d m, 2H), 2.85, 3.10 (dd, 2H), 3.37 (t, 2H). HRMS calc. forC₁₀H₂₂N₃O₂S: 248.1433 [M+H⁺], found 248.1450.

EXAMPLE 12

2-[[[(1R)-2-[(1-Iminoethyl)amino]-1-methylethyl]thio]methyl]-D-valine,dihydrochloride

The procedures and methods used in this synthesis are the same as thoseused in Example 3 except that isopropyl triflate (prepared inExample-11A) is used instead of methyl iodide to give the titlecompound.

EXAMPLE 13

2-[[[(1R/S)-2-(1-iminoethyl)amino)-1-ethylethyl]-thio]methyl]-D-valine,dihydrochloride

The procedures and methods used in this synthesis are the same as thoseused in Example 5, except that (2R)-2-isopropyl triflate (prepared inExample-11A) is used instead of methyl iodide, to give the titlecompound.

EXAMPLE 14

2-[[[(1S)-2-[(1-Iminoethyl)amino]-1-fluoromethylethyl]thio]methyl]-D-valine,dihydrochloride

The procedures and methods used in this synthesis are the same as thoseused in Example 6, except that (2R)-2-isopropyl triflate (prepared inExample-11A) is used instead of methyl iodide, to give the titlecompound.

EXAMPLE 15

S-[(R/S)-2-[(1-iminoethyl)amino]-1-(trifluoromethyl)ethyl]-2-methyl-L-cysteine,dihydrochloride

t-Butyl-N-(2-oxoethyl)carbamate is treated with 1,1,1-trifluoroethylmagnesium bromide to give(R/S)-1-[(1,1-dimethylethoxycarbonyl)]amino-4,4,4-trifluoro-2-butanol.The procedures and methods used in this synthesis are the same as thoselisted in Example 3, except that(R/S)-1-[(1,1-dimethylethoxy)carbonyl]amino-4,4,4-trifluoro-2-butanol isused instead of (S)-1-[(benzyloxycarbonyl)amino]-2-propanol, to give thetitle compound.

EXAMPLE 16

S-[2-(1-Iminoethylamino)ethyl]-2-methyl-(D/L)-cysteine,bistrifluoroacetate Example-16A S-(2-aminoethyl)-L-cysteine, methylester

A 10 g (50 mmol) sample of S-(2-aminoethyl)-L-cysteine was dissolved in400 mL of methanol. Into this cooled solution was bubbled in anhydrousHCl for 30 minutes. After stirring at room temperature overnight, thesolution was concentrated to afford 12.7 g of the title compound.

Example-16BN-{4-chlorophenyl)methylene]-S-[2-[[(4-chlorophenyl)methylene]amino]ethyl]-L-cysteine,methyl ester

A 12.7 g (50 mmol) sample of the product of Example-16A,S-(2-aminoethyl)-L-cysteine methyl ester, was dissolved in acetonitrile.To this solution was added 12.2 g (100 mmol) of anhydrous MgSO₄, 14 g(100 mmol) of 4-chlorobenzaldehyde and 100 mmol of triethylamine. Thismixture was stirred for 12 hours, concentrated to a small volume anddiluted with 500 mL of ethyl acetate. The organic solution was washedsuccessively with (0.1%) NaHCO₃, (2N) NaOH, and brine solution. Theorganic was dried (anhy. MgSO₄), filtered and concentrated to afford 7.5g of the title compound. [M+H⁺]=179.

Example-16CN-[4-chlorophenyl)methylene]-S-[2-[[(4-chlorophenyl)methylene]amino]ethyl]-2-methyl-D/L-cysteinemethyl ester

A sample of the product of Example-16B,N-{4-chlorophenyl)methylene]-S-[2-[[(4-chlorophenyl)methylene]amino]ethyl]-L-cysteinemethyl ester (7.5 g, 17 mmol), in anhydrous THF was treated with 17 mmolof sodium bis(trimethylsilyl)amide at −78° C. under nitrogen, followedby 2.4 g (17 mmol) of methyl iodide. The solution was held at −78° C.for 4 hr and then warmed to room temperature with continuous stirring.The solvents were evaporated in vacuo and brine and ethyl acetate wasadded. The aqueous phase was extracted 3×EtOAc, and the combined organiclayers were washed with 10% KHSO₄, water, and brine before it was dried(anhy. MgSO₄), filtered, and evaporated to afford the title compound.

Example-16D S-(2-aminoethyl)-2-methyl-D/L-cysteine, hydrochloride

A sample of the product of Example-16C,N-[4-chlorophenyl)methylene]-S-[2-[[(4-chlorophenyl)methylene]amino]ethyl]-2-methyl-D/L-cysteinemethyl ester (4.37 g, 10 mmol), was stirred and heated (60° C.) with 2NHCl overnight and the solution washed (3×) with ethyl acetate. Theaqueous solution was freeze-dried to give the title compound.

A sample of the product of Example-16D,S-(2-aminoethyl)-2-methyl-D/L-cysteine dihydrochloride (2.5 g (10 mmol),was dissolved in H₂O and the pH was adjusted to 10 with 1 N NaOH. Ethylacetimidate hydrochloride (1.24 g, 10.0 mmol) was then added to thereaction mixture. The reaction was stirred 15-30 min, the pH was raisedto 10, and this process repeated 3 times. The pH was reduced to 4 withHCl solution and the solution evaporated. The residue was purified onreverse phase HPLC with H₂O containing 0.05% trifluoroacetic acid as themobile phase to afford the Example 16 title product. M+H=220.

EXAMPLE 17

S-[2-[(1-Iminoethyl)amino]ethyl]-2-fluoromethyl-L-cysteine,dihydrochloride

Epibromohydrin is treated with HF-pyridine to give the dihalogenatedalcohol, which is oxidized with K₂Cr₂O₇ to give the1-bromo-3-fluoroacetone. This product is treated with(1,1-dimethylethoxy)-N-(2-sulfanylethyl)carboxamide in the presence ofNaOH to give(1,1-dimethylethoxy)-N-[2-(3-fluoro-2-oxopropylthio)ethyl]carboxamide.This is cyclized to the racemic hydantoin by NaCN and (NH₄)₂CO₃ inrefluxing ethanol and the enantiomers separated by chiralchromatography. The S-enantiomer is treated with hot 48% HBr solution toafford S-(2-aminoethyl)-2-fluoromethyl-L-cysteine dihydrochloride, whichis converted to the title compound by treatment with ethyl acetimidatein the presence of base.

EXAMPLE 18

(2R)-2-Amino-3[[2-[(1-iminoethyl)amino]ethyl]sulfinyl]-2-methylpropanoicacid, dihydrochloride

A solution of S-[2-[(1-iminoethyl)amino]ethyl]-2-methyl-L-cysteine,dihydrochloride (Example 1, 0.2 g, 0.73 mmol) in 3 mL of water wasstirred and cooled to 0° C. and a solution of 3% H₂O₂ (0.8 mL, 0.73mmol) in formic acid (0.4 mL, 0.73 mmol) was added in 0.3 mL portions.The cold bath was removed and the reaction mixture was stirred at roomtemperature for 48 hours. The solution was concentrated in vacuo,diluted with of water (10 mL) and concentrated again to give the crudesulfone. This residue was chromatographed (C-18 reverse phase, withmobile phase H₂O containing 0.05% trifluoroacetic acid) to give the puresulfone. The sulfone was treated with 1M HCl (10 mL) and concentrated invacuo to give 140 mg of a mixture of 2 diastereomers of the titlecompound as a colorless oil of the HCl salts. ¹H NMR (300 MHz, D₂O) δ1.5 (s, 2H), 1.6 (s, 1H), 2.0 (s, 3H), 3.1 (m, 2H), 3.3 (m, 2H) 3.6 (m,2H). HRMS calc. for C₈H₁₈N₃O₃S: 236.1069 [M+H⁺], found: 236.1024.

EXAMPLE 19

(2R)-2-Amino-3[[2-[(1-iminoethyl)amino]ethyl]sulfonyl]-2-methylpropanoicacid dihydrochloride

A solution of S-[2-[(1-Iminoethyl)amino]ethyl]-2-methyl-L-cysteinedihydrochloride, the product of Example 1, (0.15 g, 0.54 mmol) in 2 mLof water was cooled to 0° C. and a solution of 3% H₂O₂ (1.6 mL, 1.46mmol) in formic acid (0.8 mL, 14.6 mmol) was added. The cold bath wasremoved and the reaction mixture was stirred at room temperature for 18hours. The solution was concentrated in vacuo, diluted with 10 mL ofwater and concentrated again to give the crude sulfoxide. The residuewas diluted with 4 mL of water and was adjusted to pH 9 with 2.5 N NaOH.Acetone (5 mL) was added, followed by Boc₂O (0.2 g), and the reactionwas stirred for 48 h at room temperature. The reaction mixture wasadjusted to pH 6 with 1M HCl and was concentrated in vacuo. This residuewas chromatographed (C-18 reverse phase; 40 to 50% ACN: H₂O, 0.05% TFA)to give the pure Boc protected material. The fractions were concentratedin vacuo and the residue was treated with 1N HCl (3 mL) for 1 h. Thesolution was concentrated to give 30 mg of the title compound ascolorless oil. ¹H NMR (400 MHz, D₂O) δ 4.0 (d, 1H), 3.7 (d, 1H), 3.6 (t,2H), 3.5 (t, 2H), 2.1 (s, 3H), and 1.5 (s, 3H) ppm. HRMS calc. forC₈H₁₈N₃O₄S: 252.1018 [M+H⁺], found: 252. 0992.

EXAMPLE 20

S-[2-[(1-Iminoethyl)amino]ethyl]-2-methyl-L-cysteine

DOWEX 50WX4-200 (250 g) in a glass chromatography column (38×560 mm) waswashed with water until the eluent was at pH 6. An solution of theproduct of Example 1,S-[2-[(1-Iminoethyl)amino]ethyl]-2-methyl-L-cysteine dihydrochloride (6g) dissolved in water was placed on the column, which was washed withwater until the pH returned to 6. The column was then washed with 0.07 MNH₄OH (flow rate 15 mL/min) and the basic fractions were immediatelyplaced in a dry ice/acetone bath. The fractions were pooled andconcentrated to dryness by lyophilization to give the title compound. ¹HNMR (400 MHz, DMSO-d₆) δ 3.4 (m, 1H), 3.3 (m, 1H), 3.0 (d, 1H), 2.7 (m,11H), 2.4 (m, 1H), 2.1 (s, 3H), and 1.1 (s, 3H).

Analysis calculated for C₈H₁₇N₃O₂S+0.6H₂O: C, 41.76; H, 7.97; N, 18.26;found C, 41.43; H, 7.47; N, 17.96; Cl, trace.

EXAMPLE 21

S-[2-[(1-Iminoethyl)amino]ethyl]-2-methyl-L-cysteine, dihydrochlorideExample-21A(2R,4R)-Methyl-2-tert-butyl-1,3-thiazoline-3-formyl-4-carboxylate

The title material was prepared according to J. Chem. Soc. Perkin Trans.1991 p 2291 and Tetrahedron 1993 p 2131. To a 2 L RB flask fitted with areflux condenser, a Dean-Stark trap, an overhead stirrer and athermocouple was added pivalaldehyde (23.7 g, 0.275 mole) dissolved in700 mL of toluene. Agitation was started and L-cysteine hydrochloridemethyl ester (45 g, 0.262 mole) was added to the stirring solution.Triethylamine (29.2 g, 0.288 moles) was then added to the batch in astream over a few minutes. The reaction mixture was heated to reflux andwater was removed. The batch was heated for a total of 3h, cooled andfiltered. The salt cake was washed with 250 mL of fresh toluene and thewash was combined. Formic acid (24.1 g, 0.524 moles) and solid sodiumformate (19.6 g, 0.288 moles) were then added and the resultingsuspension was cooled to −5° C. Acetic anhydride (53.5 g, 0.524 moles)was carefully added to the mixture keeping the batch temperature below5° C. After the addition, the reaction was allowed to warm to roomtemperature and stirring continued for 18h during which time the productprecipitated. The crude product was filtered and redissolved in 400 mLof EtOAc and filtered to remove insoluble sodium salts. The organicsolution was then neutralized with 200 mL of saturated sodiumbicarbonate solution and the final aqueous layer was near pH 7. Theorganic layer was separated and the aqueous layer was extracted withethyl acetate. The organic layers were combined and concentrated to givethe crude product (60.2 g) as a viscous oil which slowly crystallized toa white solid. The solid was washed with cyclohexane containing 4% of2-propanol to give 41.01 g of the title product in >99.5% purity and67.8% yield as determined by GC. The desired title product cis isomerwas present in over 98%.

Example-21B(2R,4R)-Methyl-2-tert-butyl-1,3-thiazoline-3-formyl-4-methyl-4-carboxylate

Anhydrous lithium chloride (43.0 g, 0.102 moles) was mixed with 300 mLof dimethoxyethane and 500 mL of THF until a clear solution wasobtained. A THF solution of the product of Example-21A (50.0 g, 0.216moles) was added and cooled to −65° C. under a nitrogen atmosphere.Iodomethane (45.0 g, 0.316 moles) diluted with 45 mL of THF was addedfollowed by 230 mL of 1.0M THF solution of lithiumbis-trimethylsilylamide. The reaction was stirred at −65° C. for 10 h.The batch was quenched with 30 g of acetic acid in 600 mL water andextracted with 500 mL of ethyl acetate. The organic layer was washedwith saturated sodium bicarbonate and concentrated to give 51.22 g (96%)of the title product as a light brown solid.

Example-21C (2R) 2-Methyl-L-cysteine hydrochloride

A sample of the product of Example-21B (20 g, 83 mmoles) was placed in around bottom flask equipped with an overhead stirrer and refluxcondenser. To this solid was added 100 mL of concentrated hydrochloricacid. The reaction was slowly heated to 95° C. for 7 days. The reactionwas then treated with 250 mL of toluene to remove non-polar organicimpurities. The aqueous solution was then concentrated. The crude titleproduct was obtained as an orange resin weighing 14 g. The resin waspowdered up under ethylether/methylene chloride and filtered to give 13g of the title material as a pale brown hygroscopic powder. ¹H NMR (D₂O)δ 4.70 (s, HDO exchange), 3.08 (d, 1H), 2.80 (d, 1H), 1.48 (s, 3H).

Example-21D 2-[(1,1-dimethylethoxycarbonyl)-amino]ethyl bromide

A 5L RB flask equipped with overhead stirrer, thermocouple and nitrogeninlet was charged with 3 liters of ethyl acetate and agitation started.To this was added di-tert-butyldicarbonate(545 g, 2.50 moles) and2-bromoethylamine hydrobromide (553.7 g, 2.70 moles) under a nitrogenblanket. The batch was cooled to 5° C. in an ice bath andN-methylmorpholine, (273 g, 2.70 moles) was added dropwise over ca. 0.5h. After the addition was complete, the batch was allowed to stirovernight and warm to ambient temperature. After 16h, the batch wasquenched by adding 1.5L of DI water. The organic layer was washed withdilute HCl, sodium bicarbonate solution followed by brine. The driedorganic solution had solvents removed to give an oil that froze to alight yellow solid. A total of 496 g (88% yield) of the title productwas obtained in approx. 96% purity.

Example-21ES-[2-[[(1,1-dimethylethoxy)carbonyl]amino]ethyl]-2-methyl-L-cysteineacetate

A 3 L flask, charged with 435 g of methanol, was equipped with anoverhead stirrer, a thermocouple, and maintained under a N₂ purge. Tothe reaction flask was added 150.0 g (0.804 moles) of the product ofExample-21C carefully with stirring to dissolve. A solution of KOHprepared by dissolving 154.7 g of solid KOH in 840 mL of degassedmethanol was added to the reaction solution dropwise keeping thetemperature between 20-30° C. The product of Example-21C (180.2 g, 0.804moles) was dissolved in 375 mL of methanol and this solution was addeddropwise to the cold reaction mixture over 1 hour at 10-12° C. When thereaction was complete, the batch had its pH adjusted to pH 5. Thereaction mixture was then filtered through a pad of Celite and thefiltrate was concentrated to yield 454 g of a tan solid product. Thistitle product was slurried with ethyl acetate to give an off-white solidweighing 299 g. The crude title product solid was carried on to the nextstep without further purification. ¹H NMR as acetate (D₂O) δ 4.68 (s,D₂O exchange), 3.12 (m, 3H), 2.68 (m, 3H), 1.83 (s, 3H), 1.42 (s, 3H),1.32 (s, 9H).

Example-21F S-(2-aminoethyl)-2-methyl-L-cysteine

To a round bottom flask equipped with an overhead stirrer and nitrogenpurge was added 150 mL of 37% hydrochloric acid. The agitator wasstarted and 150 mL of water was added to the vessel followed by 173 g ofcrude product of Example-21E. The reaction was stirred for two hours andthe clear brown solution batch was concentrated to give the crude di-HClsalt of the title product as a brown syrup (ca.157 g). This wasredissolved in 200 mL of water and decolorized with charcoal. Thesolution was passed through a Dowex resin column and the neutral titleproduct was eluted with aqueous ammonium hydroxide to yield 64% of thismaterial with approx. 94% purity. ¹H NMR (D₂O, 300 MHz) δ 4.68 (s, D₂Oexchange), 2.9 (m, 3H), 2.6 (m, 3H), 1.20 (s, 3H).

Polymer bound triazabicyclo[4.4.0] dec-5-ene resin (Fluka), 38 g, wassuspended in 160 mL of ethanol in a round bottom flask. The amino acidproduct of Example-21F (7.5 g) in 40 mL of ethanol was added to thestirring resin slurry. Ethyl acetimidate, 6.5 g (53 mmoles) was addedportion-wise to the reaction. The reaction was stirred under nitrogenfor 16h. The resin was filtered and washed on the filter with 100 mL ofethanol containing 10 mL of conc. HCl. The combined filtrate wasconcentrated to give 12 g of crude title product as a pale brown viscoussemisolid. The yield was approximately 60-70% and the title productshowed 90% purity. The title product was further purified by reversephase chromatography. ¹H NMR (D₂O) δ 4.74 (s, D₂O exchange), 3.37 (t,2H), 3.08 (d, 1H), 2.93 (d, 1H), 2.74 (m, 2H), 2.06 (s, 3H), 1.48 (s,3H).

EXAMPLE 22

S-[2-[(1-Iminoethyl)amino]ethyl]-2-methyl-L-cysteine, dihydrochlorideExample-22A N-Boc-cysteamine

A 3L 4-neck RB flask was purged with nitrogen for 20 min and thencharged sequentially with 2-aminoethanethiol hydrochloride (113.6 g, 1mol), di-tert-butyl-dicarbonate (218.3 g, 1 mol) and 500 mL of toluene.The mixture was cooled with an ice-water bath and purged with nitrogenfor 10 min. Sodium hydroxide (2.5N, 880 mL, 2.2 mol) was added to thestirring mixture in about 1.5 h at between 0 and 11° C. After theaddition of sodium hydroxide was complete, the cooling bath was removedand the resulting reaction mixture was allowed to warm up to roomtemperature and stirred at ambient temperature overnight. This provideda solution of the title product.

Example-22B

The product solution of Example-22A was cooled with an ice-water bath. Asample of chloroacetone (101.8 g, 1.1 mol) was added to the vigorouslystirred reaction mixture over about 50 min at between 8 and 11° C. Afterthe addition of chloroacetone was completed, the cooling bath wasremoved and the resulting reaction mixture was allowed to stir at roomtemperature overnight. The toluene layer was separated, washed withwater (250 mL) and concentrated on a rotary evaporator at 85° C. underhouse vacuum followed by high vacuum to give the crude titled compound(225.7 g, 96.7%). ¹H NMR (CDCl₃, 400 MHz) δ 4.95 (bs, 1H), 3.20 (m, 4H),2.54 (t, 2H), 2.20 (s, 3H), 1.35 (s, 9H).

Example-22C[2-[[(4-Methyl-2,5-dioxo-4-imidazolidinyl)methyl]thio]ethyl]carbamicacid, 1,1-dimethylethyl ester

To a 3L 4-neck RB flask equipped with an overhead stirrer, athermocouple and a condenser connected to an empty flask and a caustictrap, was added the product of Example-22B (70 g, 0.3 mol), absoluteethanol (80 mL), sodium cyanide (19.1 g, 0.39 mol), ammonium carbonate(43.3 g, 0.45 mol) and water (720 mL) in this order. The 4^(th) neck wasclosed with a stopper. The resulting reaction mixture was heated atbetween 67 and 68° C. for 6 h. Subsequently, the almost clear brownsolution was cooled to room temperature. Upon cooling, solid began toform and the heterogeneous mixture was stirred at room temperatureovernight. The reaction mixture was then acidified with 12% hydrochloricacid to pH 2 in about 1 h at between −2 and 2° C. The cold reactionmixture was stirred at pH2 for additional 30 min and then filtered. Theflask was rinsed with distilled water (2×250 mL) and each rinse was usedto wash the solid cake. The solid was again washed with distilled water(2×250 mL) and then air-dried for 4 days. The dry solid was trituratedwith 200 mL of toluene for 0.5 h. The slurry was filtered. The solid wasrinsed sequentially with toluene (50 mL) and 1:4 ratio of toluene/hexane(100 mL) and then air-dried at room temperature overnight to give 83.1%yield of the titled compound, m.p. 134-136° C. ¹H NMR (DMSO_(d6), 400MHz) δ 10.62 (s, 1H), 7.85 (s, 1H), 6.83 (m, 0.9H), 6.48 (bs, 0.1H),3.29 (s, 2H), 2.99 (m, 2H), 2.71 (s, 2H), 2.95 (m, 2H), 1.32 (s, 9H),1.24 (s 3H); ¹³C NMR (DMSO_(d6), 400 MHz), δ 178.1, 157.1, 156.1, 78.4,63.7, 40.7, 39.4, 33.2, 28.9, 23.8. Analysis Calcd for C₁₂H₂₁N₃O₄S: C,47.51; H, 6.98; N, 13.85; S, 10.57. Found: C, 47.76; H, 6.88; N, 13.77;S, 10.75.

Example-22D R andS-[2-[[(4-Methyl-2,5-dioxo-4-imidazolidinyl)methyl]thio]ethyl]carbamicacid, 1,1-dimethylethyl ester

The reaction product of Example-22C was separated into its R and Senantiomers on a Chiralpak® AD column eluting with methanol. The Sisomer was the first eluting isomer followed by its R enantiomer. Bothisomers were used in subsequent transformations.

S Enantiomer:

[α]in MeOH at 25° C.=+43.0 (365 nm). ¹HNMR: (400 mHz, CD₃OD) δ 1.49 (s,9H), 2.05 (s, 3H), 2.65 (t, 2H), 2.9 (q, 2H, d), 3.20 (m, 2H). IR:λcm⁻¹=1772, 1709.

Analysis calculated for C₁₂H₂₁N₃O₄S (formula weight=303.38): C, 47.51;H, 6.98; N, 13.85. Found: C, 47.39; H, 6.62; N, 13.83. M+H=304.

R Enantiomer:

[α]in MeOH at 25° C.=−46.3 (365 nm). ¹HNMR: (400 mHz, CD₃OD) δ 1.48 (s,9H), 2.05 (s, 3H), 2.65 (t, 2H), 2.85 (q, 2H, d), 3.18 (m, 2H). IR:?cm⁻¹=1770, 1711.

Analysis calculated for C₁₂H₂₁N₃O₄S (formula weight=303.38): C, 47.51;H, 6.98; N, 13.85. Found: C, 48.15; H, 7.04; N, 14.37. M+H=304.

Example-22E S-(2-aminoethyl)-2-methyl-L-cysteine

Acid Hydrolysis Method:

A 500 mL three-necked round bottom flask equipped with a distillationcondenser was charged with the R-isomer product of Example-22D (45.8 g,150.9 mmol) and treated portion wise with 48% aq. HBr (160 mL) at roomtemperature with stirring. After the gas evolution ceased, the mixturewas heated with a heating mantle until the pot temperature reached to126° C. while the volatile t-butyl bromide (bp 72-74° C.) followed by asmall amount of aq. HBr (approx. 15 mL) were distilled off. Thedistillation condenser was replaced with a reflux condenser and themixture was heated at reflux for 30 hours. The solution was concentratedand the residue was dissolved in water (250 mL) and loaded on to aDowex® 50WX4-200 ion-exchange resin (8.5×11 cm) and eluted with water(2L) followed by dilute aqueous ammonium hydroxide (30 mL of 28-30%ammonium hydroxide diluted to 1000 mL with water, 3L). Fractionscontaining the desired product were combined, concentrated, and driedunder vacuum at 75-80° C. for two hours to give 22.1 g (82%) of thetitle product, S-(2-aminoethyl)-2-methyl-L-cysteine, as a white solid.Proton and C-13 NMR spectra are consistent with the title product. Mp157° C. ¹H NMR (400 MHz, D₂O) δ 1.19 (3H, s), 2.53 (1H, d, J=13.6 Hz),2.57-2.72 (2H, nm), 2.92 (1H, d, J=13.6 Hz), 2.92 (2H, t, J=6.8 Hz); ¹³CNMR (100 MHz, D₂O) δ 24.7, 31.3, 38.9, 40.9, 59.6, 180.7.

Analysis Cald for C₆H₁₄N₂O₂S+0.1H₂O: C, 40.02; H, 7.95; N, 15.56; S,17.81. Found: C, 39.93; H, 7.98; N, 15.38; S, 17.70.

Base Hydrolysis Method:

To a stainless steel autoclave equipped with agitation was added 24.2 g(0.08 moles) of the R-isomer product of Example-22D. After purging theapparatus with nitrogen, 128 g (0.32 moles) of 10% caustic was addedgenerating a solution. The autoclave was sealed and heated to 120° C.for 30 hours. After cooling to room temperature, the autoclave wasvented to give 142 ml (151 g) of an aqueous solution of the sodium saltof the title product. H¹NMR (sample acidified with HCl and diluted withD₂O, 400 MHz): δ 1.47 (s,3H), 2.75 (m, 2H), 2.90 (d,1H, J=14.8 Hz), 3.06(t, 2H, J=6.4 Hz), 3.14 (d, 1H, J=14.8 Hz). C¹³NMR (sample acidifiedwith HCl and diluted with D₂O, 100 MHz): δ 172.9, 60.8, 39.1, 39.0,30.4, 22.2. MS (MS/CI-LC) M+1179.

DBU (218 μL; 1.46 mmol) and ethyl acetimidate hydrochloride (171 mg;1.34 mmol) were dissolved in ethanol (6 mL) in a 25 mL, one-necked,round-bottomed flask at room temperature (˜20° C.). The title product ofExample-22E (200 mg; 1.12 mmol) was added in one portion to thissolution. The mixture was stirred until the title product of Example-22Ewas consumed (1-2 hours). The mixture was chilled with an ice-bath andthen treated with 6 M HCl (830 μL). ¹HNMR analysis indicated a chemicalyield of 95 mole % or better. The solvent was evaporated and the titleproduct of Example-22 was purified by reverse-phase or ion-exchangechromatography.

A 210 gm solution (containing ˜20 g of the title product of Example-22Eof the base hydrolysis reaction product was put into a 500 mL,three-necked, round-bottomed flask. The apparatus was equipped with amechanical stirrer, a Dean-Stark apparatus (20 mL with a stopcock), acondenser, and a temperature controller. Water (140 mL) was distilledoff from the mixture. 1-butanol (150 mL) was added to the pot and theremaining water (37 mL) was distilled azeotropically. Additional1-butanol (13 mL) was removed by distillation until the pot temperaturereached 117° C. The butanol slurry was cooled to room temperature andfiltered through a pad of celite. The salts were washed with l-butanol(2×20 mL). DBU (21.8 μL; 146 mmol) and ethyl acetimidate hydrochloride(17.1 mg; 134 mmol) were dissolved in 1-butanol (40 mL) in a 500 mL,three-necked, round-bottomed flask at room temperature. The apparatuswas equipped with a mechanical stirrer, an addition funnel, and atemperature probe. The title product of Example-22E/1-butanol solutionwas put into the addition funnel and added to the ethyl acetimidate/DBUsolution while maintaining the pot temperature below 25° C. The mixturewas stirred until the starting material was consumed (2-3 hours). Asolution of conc. HCl (94 mL) and water (100 mL) was put into a 1 L,three-necked, round-bottomed flask and chilled to 0° C. The apparatuswas equipped with a mechanical stirrer, an addition funnel, and atemperature probe. The reaction mixture was put into the additionfunnel. The reaction mixture was added to the aqueous HCl solution whilemaintaining the temperature below 25° C. Ethyl acetate (100 mL) wasadded to the solution and the layers were separated. The aqueous layerwas washed once more with ethyl acetate (100 mL). ¹HNMR analysisindicated a chemical yield of 95 mole % or better. This title product ofExample-22 was purified by reverse-phase or ion-exchange chromatography.

¹HNMR (400 MHz, D₂O) δ 1.49 (3H, s), 2.08 (3H, s), 2.74 (2H, m), 2.91(1H, d), 3.17 (1H, d), 3.35 (2H, t).

EXAMPLE 23

S-[2-[(1-Iminoethyl)amino]ethyl]-2-methyl-L-cysteine methyl ester,dihydrochloride

The product of Example 22 (1.0 g, 3.42 mmol) dissolved in anhydrousmethanol (40 ml) was added to a 500 ml 3 neck round bottom flaskequipped with a magnetic stirrer and a thermocouple. This reaction undernitrogen was cooled to 0° C. HCl gas was bubbled into the reactionsolution for 1 minute. The reaction mixture was allowed to warm to roomtemperature and continued to stir overnight. A sample was taken from thereaction mixture and concentrated. NMR and Mass Spectrometry indicatedstarting material and product. The solvent was stripped and the oilyresidue was re-dissolved in anhydrous methanol (40 ml), cooled to 0° C.,and HCl gas was bubbled into the solution for 1 minute. The reactionmixture was allowed to warm to room temperature and stir overnight. Asample was taken from the reaction mixture and concentrated. NMR andMass Spectrometry indicated minimal starting material and majorityproduct. The solvent was stripped and the oily residue was re-dissolvedin anhydrous methanol (40 ml), cooled to 0° C., and HCl gas was againbubbled into the solution for 1 minute. The reaction mixture was allowedto warm to room temperature and stir overnight. A sample was taken fromthe reaction mixture and concentrated. NMR and Mass Spectrometryindicated only the desired title product. The reaction mixture wasconcentrated to provide 1.01 g of a light yellow oil, 97% yield. Thereaction mixture was stirred in acetonitrile (50 ml) for 3 hours and thetitle product as a white fine powder was recovered, 484 mg. MassSpectrometry: (ZMD Waters Micromass, Electrospray), M+H at 234.2

¹HNMR: (400 mHz, D₂O) δ 1.51 (s, 3H), 2.09 (s, 3H), 2.72 (t, 2H), 2.97(d, 1H), 3.19 (d, 1H), 3.36 (t, 2H), 3.73 (s, 3H). ¹³CNMR: δ 18.58,21.69, 30.79, 37.79, 41.58, 54.24, 60.75, 165.41, 171.35.

Analysis calculated for C₉H₁₉N₃O₂S+2 HCl+0.3H₂O (311.66): C, 34.68; H,6.98; N, 13.48; Cl, 22.75; S, 10.29. Found: C, 34.51; H, 6.99; N, 13.75,Cl, 22.75, S, 10.43.

EXAMPLE 24

2-methyl-S-[2-(3-methyl-5-oxo-1,2,4-oxadiazol-4(5H)-yl)ethyl]-L-cysteine, monotrifluoroacetate Example-24AN′-hydroxyethanimidamide

To a 3L round bottom flask was added hydroxylamine hydrochloride (138.98g, 2.0 mol) in ethanol (1.2L) followed by a slow addition of sodiumethoxide (136.1 g, 2.0 mol). The temperature was maintained between 25°C. and 30° C. The reaction was then stirred for 30 minutes at roomtemperature. The precipitated NaCl was filtered off and washed withethanol (100 mL). The hydroxylamine free base in the filtrate was addedto a 3L flask and acetonitrile (112.75 g, 2.75 mol). This mixture wasthen subjected to reflux over night. After cooling, the solvent wascarefully removed in vacuo to 50% of its original volume. The reactionthen sat in an ice bath for one hour were upon crystals formed and werefiltered off. The filtrate was carefully concentrated to 50% of itsvolume again. The reaction was placed into ice and the resultingcrystals were again isolated by filtration to afford 52 g (35%) of thetitle product.

Example-24B Potassium 3-methyl-1,2,4-oxadiazolin-5-onate

To a 25 mL round bottom flask was added the product of Example-24A (1 g,0.013 mol), potassium t-butoxide (1.59 g, 0.013 mol) and diethylcarbonate (8.18 mL. 0.067 mol). The reaction was then refluxed for 5hours. The solvent was removed and the resulting solid was trituratedwith methylene chloride and diethyl ether. The solid title product wasthen dried under high vacuum to afford 1.57 g (87%). ¹H NMR (d₆-DMSO,300 MHz) δ 1.69 (bs, 3H). ³C NMR (d₆-DMSO, 99 MHz) δ 13.26, 166.54,173.99.

Example-24C 3-methyl-1-(1-bromoethyl)-2,4-oxadiazolin-5-one

To a 250 mL Round Bottom Flask was added the title product ofExample-24B, potassium 3-methyl-1,2,4-oxadiazolin-5-onate, (10.13 g,0.0734 mol) in DMF (100 mL). To the slurry was added 1,2-dibromo ethane(31.54 mL, 0.366 mol). The reaction was heated in an oil bath for 2hours at 130° C. The oil bath was removed and the reaction cooled afterwhich water (200 mL) and ethyl acetate (50 mL) were added. The organicswere collected and washed 3×100 mL brine. The organics were dried overMgSO₄ and then concentrated in vacuo to afford 9.1 g (60%) of the titlecompound. ¹H NMR (CDCl₃, 300 MHz) δ 2.21 (s, 3H), 3.12 (t, 2H), 3.91 (t,2H).

The 75 mL of methanol in a 100 mL round bottom flask was deoxygenated bybubbling nitrogen through it for 5 minutes. To 50 mL of this methanolwas added NaOH (1.6 g, 0.040 mol). The suspension was stirred in an oilbath at 45° C. for 30 minutes after which the NaOH had dissolved. Theresulting solution was cooled to room temperature and alpha-methylcysteine (1.72 g, 0.010 mol) was added in 10 mL of deoxygenatedmethanol. The reaction stirred for 45 minutes at room temperature. Tothis reaction was added the product of Example-24C (2.07 g, 0.010 mol)in 10 mL of deoxygenated methanol. The reaction was complete asindicated by mass spectral analysis after it had been stirred overnight. The reaction mixture was diluted with water (100 mL) and purifiedusing reverse phase chromatography to afford 3.0 g (93%) of the titleproduct of Example 24 as its trifluoroacetate salt. M.S. M+H⁺(262.0),M+Na⁺(282.0). ¹H NMR (CD₃OD, 300 MHz) δ 1.39 (s, 3H), 2.23 (s, 3H), 2.74(m, 2H), 2.84 (m, 2H), 3.72 (t, 2H).

EXAMPLE 25

[2-[[[(4R)-4-methyl-2,5-dioxo-4-imidazolidinyl]methyl]thio]ethyl](1-N-iminoethyl)amine

The[2-[[[(4R)-4-methyl-1-2,5-dioxo-4-imidazolidinyl]methyl]thio]ethyl]carbamicacid, 1,1-dimethylethyl ester isomer product of Example-22D (2.05 g, 6.5mmol) was dissolved in 25 mL 4.0 N HCl in Dioxane and stirred for 10min. After the addition of 2N HCl (5 mL) the reaction was stirred foradditional 2 h. The reaction mixture was then concentrated under reducedpressure to give 1.68 g of a red brown gummy solid. This material wastaken up in 25 mL of deionized water and the pH was adjusted to 8.4 with2N NaOH. Ethylacetimidate hydrochloride (2.39 g, 0.019 mol) was thenadded while maintaining the pH at 8.4. The reaction mixture was thenstirred at room temperature for one hour at pH 8.4. The pH of thereaction mixture was then adjusted to 3.5 by adding an appropriateamount of 1 N HCl and stirred for another 16 h. The reaction mixture wasthen concentrated on a rotary evaporator to obtain the crude productthat was purified on a Gilson preparative HPLC to give the desiredproduct as a white hygroscopic solid in 70% yield.

Mass M⁺¹=245. [α]in H₂O at 25° C.=−37.6 (365 nm).

Analysis calculated for C₉H₁₆N₄O₂S+1.0 HCl+1.3H₂O (formulaweight=304.20): C, 35.54; H, 6.49; N, 18.42; Cl, 11.65; S, 10.54. Found:C, 35.83; H, 6.08; N, 18.55, Cl, 11.19, S 10.63.

EXAMPLE 26

[2-[[[(4S)-4-methyl-2,5-dioxo-4-imidazolidinyl]methyl]thio]ethyl](1-N-iminoethyl)aminehydrochloride Example-26A(5S)-5-[[(2-aminoethyl)thio]methyl]-5-methyl-2,4-imidazolidinedione,monohydrochloride

The[2-[[[(4S)-4-methyl-2,5-dioxo-4-imidazolidinyl]methyl]thio]ethyl]carbamicacid, 1,1-dimethylethyl ester isomer product of Example 22D was purifiedby chromatography using 66% Ethyl Acetate in Toluene, Biotage Flash 75silica gel. A sample of this material (5.9 g, 16.5 mmol, [α]in MeOH at25° C.=+45.7, 365 nm) was then dissolved in 165 mL THF and treated with4.125 mL 4.0 N HCl in Dioxane. The reaction was allowed to stir for twohours at room temperature and monitored by TLC. The free amine productwas then purified by chromatography using reverse phase media(YMC-ODS-AQ) to yield 4.8 g of the title material.

A 3.5 g (17.2 mmol) sample of the product of Example-26A was treatedwith 10% NaOH solution to pH 9-10. To this solution was added 4.26 g ofethyl acetimidate hydrochloride while adjusting pH to 9 by adding asolution of 10% NaOH. After stirring at pH 9 for 2 hours, the pH wasadjusted to 7.5 by adding an appropriate amount of 0.1 N HCl. Thissolution was stirred for another 2 hours before the pH was furtheradjusted to 4.5 by adding 0.1 N HCl. After stirring this solution for 10hours the water was removed under reduced pressure (11 mbar) and 47° C.water bath. The crude title product was chromatographed using reversephase media (YMC-ODS-AQ) to give 156 mg of the title material. [α]in H₂Oat 25° C.=+54.8 (365 nm).

Analysis calculated for C₉H₁₆N₄O₂S+1.0 HCl+0.85H₂O (formulaweight=296.09): C, 36.51; H, 6.37; N, 18.92; Cl, 11.97; S, 10.83. Found:C, 36.69; H, 6.32; N, 18.85, Cl, 11.46, S 11.12.

EXAMPLE 27

N-(ethoxycarbonyl)-S-[2-[(1-iminoethyl)amino]ethyl]-2-methyl-L-cysteine,monohydrochloride

A sample of the product of Example 1 (3.22 g, 0.01 mol) was taken up in50 mL of deionized water and to this K₂CO₃ (2.76 g) was added followedby the addition of ethychloroformate (1.08 g, 0.01 mol). The reactionmixture was stirred at 25° C. for 1 hour and then concentrated on rotaryevaporator to give a white solid. This solid was purified by HPLC togive the desired product.

Mass M⁺¹=292

EXAMPLE 28

S-(2-aminoethyl)-2-methyl-D-cysteine dihydrochloride

A sample of the[2-[[[(4S)-4-methyl-2,5-dioxo-4-imidazolidinyl]methyl]thio]ethyl]carbamicacid, 1,1-dimethylethyl ester product of Example 22D (1.025 g, 3.25mmol) was dissolved in 35 mL conc. HCl and stirred for 46 h at refluxtemperature. The reaction mixture was then concentrated under reducedpressure to give 900 mg of a red brown gummy solid. This crude productwas purified by reverse phase HPLC to give pureS-(2-aminoethyl)-2-methyl-D-cysteine dihydrochloride (800 mg, 98%yield). Mass M⁺¹=179. [α]in H₂O at 25° C.=−85.6 (365 nm).

Analysis calculated for C₆H₁₄N₂O₂S+2 HCl+1H₂O+1.6 NH₄Cl (formula weight356.39; exact mass 178.07): C, 20.22; H, 7.35; N, 14.15, Cl, 35.81, S,9.00. Found: C, 20.09, H, 6.95, N, 14.55, Cl, 36.15, S, 9.56.

EXAMPLE 29

[2-(1-Iminoethylamino)ethyl]-2-methyl-D-cysteine hydrochloride

A sample of the product of Example 28 (1.25 g, 0.005 mol) was taken upin 20 mL of de-ionized water and the pH was adjusted to between 8.5 and9 with 0.1N NaOH. Ethylacetimidate hydrochloride (2.39 g, 0.019 mol) wasthen added to the stirred reaction while maintaining the pH at 8.5. Thereaction mixture was then stirred at 25° C. and 8.5 pH for 2 hours. ThepH of the reaction mixture was then adjusted to 4.0 by adding anappropriate amount of 0.1 N HCl. The reaction mixture was thenconcentrated on a rotary evaporator and the crude product residue waspurified on a Gilson HPLC system using YMC AQ column with 0.1%AcOH/CH₃CN/H₂O to give the desired product in quantitative yield. MassM⁺¹=220. [α]in H₂O at 25° C.=−134.5 (365 nm).

Analysis calculated for C₈H₁₇N₃O₂S+1.2 HCl+2H₂O (formula weight 299.09;exact mas 219.10): C, 32.13; H, 7.48; N, 14.05, Cl, 14.22, S, 10.72.Found: C, 32.39; H, 7.26; N, 14.05; Cl, 14.33; S, 10.42.

EXAMPLE 30

S-[2-[(1-Iminoethyl)amino]ethyl]-2-methyl-L-cysteine, acetate

Bio-Rad AG 1×8 resin, 200-400 mesh in the acetate form (300 g, 960meqv.) was slurried in HPLC grade water and loaded onto a column 8 cm indiameter. The water was drained to the top of the column before 37 g(116 mmol) of the product of Example 1, dissolved in 10 mL of water, wasloaded onto the column. The material was then eluted with 1 L of water.The first 200 mL fraction contained no product but the subsequent 500 mLyielded 30 g of the desired title product as a white glassy solid afterremoval of the water under reduced pressure.

Analysis calculated for C₈H₁₇N₃O₂S+CH₃COOH+1.3H₂O: C, 39.67; H, 7.86; N,13.88. Found: C, 39.96; H, 7.87; N, 13.69.

EXAMPLE 31

S-[2-[(1-Iminoethyl)amino]ethyl]-2-methyl-D-cysteine, acetate

A sample of the product of Example 29 as its mono hydrochloride salt(101 mg, 0.33 mmol) was converted to the title mono acetate by themethod of Example 30.

Analysis calculated for C₈H₁₇N₃O₂S.CH₃COOH+0.05 HCl+2.2H₂O: C, 37.41, H,8.01; N, 13.2, Cl, 0.56. Found: C, 37.30; H, 7.92; N, 13.17, Cl, 0.41.

EXAMPLE 32

S-[2-[(1-Iminoethyl)amino]ethyl]-2-methyl-L-cysteine, monohydrochloride

This material was prepared by passing the product of Example 1 through areverse phase column using the conditions described in Example 28.

Analysis calculated for C₈H₁₇N₃O₂S+1.05 HCl+0.8H₂O: C, 35.35; H, 7.36;N, 15.44; Cl, 13.65. Found: C, 35.33; H, 7.28; N, 15.45, Cl, 13.68.

EXAMPLE 33 D-galacturonic acid salt ofS-[2-[(1-Iminoethyl)aminolethyl]-2-methyl-L-cysteine

To a stirred 10 mL 0.001 M solution of acetate salt product of Example30 was added D-galacturonic acid monohydrate (0.21 g, 0.001 mole). Afterstirring for 2 hours, the solution was concentrated under vacuum. Thetitle galacturonic acid salt was dissolved in 10 mL of water andlyophilized.

EXAMPLE 34 Succinic acid salt ofS-[2-[(1-Iminoethyl)amino]ethyl]-2-methyl-D-cysteine

The title material was prepared by the method of Example 33 fromsuccinic acid and the product of Example 31.

Analysis calculated for C₈H₁₇N₃O₂S+C₄H₆O₄+1.5H₂O: C, 39.55; H, 7.19; N,11.53. Found: C, 39.24; H, 6.04; N, 11.41.

EXAMPLE 35 Succinic acid salt ofS-[2-[(1-Iminoethyl)amino]ethyl]-2-methyl-L-cysteine

The title material was prepared by the method of Example 33 fromsuccinic acid and the product of Example 30.

Analysis calculated for C₈H₁₇N₃O₂S+C₄H₆O₄+1.1H₂O: C, 39.99; H, 7.40; N,12.33. Found: C, 40.35; H, 7.11; N, 11.76.

EXAMPLE 36 Ethanolamine salt ofS-[2-[(1-Iminoethyl)amino]ethyl]-2-methyl-L-cysteine

The title material was prepared by the method of Example 33 fromethanolamine and the product of Example 30.

Analysis calculated for C₈H₁₇N₃O₂S+C₂H₇NO+2HCl+1.3H₂O: C, 31.73, H,7.67; N, 14.80; Cl, 18.73. Found: C, 31.41; H, 7.60; N, 15.00, Cl,19.12.

EXAMPLE 37 Ethylene diamine salt ofS-[2-[(1-Iminoethyl)amino]ethyl]-2-methyl-L-cysteine

The title material was prepared by the method of Example 33 fromethylene diamine and the product of Example 30.

Analysis calculated for C₈H₁₇N₃O₂S+2HCl+C₂H₈N₂+1.2H₂O: C, 32.12; H,7.92; N, 18.73, Cl, 18.96. Found: C, 31.90; H, 9.19; N, 18.08, Cl,19.11.

EXAMPLE 38 DL-Aspartatic acid salt ofS-[2-[(1-Iminoethyl)amino]ethyl]-2-methyl-L-cysteine

The title material was prepared by the method of Example 33 fromDL-aspartatic acid and the product of Example 30.

Analysis calculated for C₁₂H₂₄N₄O₆S+1.8H₂O+0.4 HOAc (formulaweight=408.86): C, 37.60; H, 7.20; N, 13.70. Found: C, 37.59; H, 7.66;N, 13.73.

EXAMPLE 39 D-Glutamic acid salt ofS-[2-[(1-Iminoethyl)amino]ethyl]-2-methyl-L-cysteine

The title material was prepared by the method of Example 33 fromD-glutamic acid and the product of Example 30.

Analysis calculated for C₁₃H₂₆N₄O₆S+1.8H₂O+0.3 HOAc (formulaweight=416.88): C, 39.18; H, 7.45; N, 13.44. Found: C, 39.47; H, 7.52;N, 13.29.

EXAMPLE 40 Citric acid salt ofS-[2-[(1-Iminoethyl)amino]ethyl]-2-methyl-L-cysteine

The title material was prepared by the method of Example 33 from citricacid and the product of Example 30.

Analysis calculated for C₁₄H₂₅N₃O₉S+0.5H₂O+0.1 HOAc+0.15 EtOH (formulaweight=416.88): C, 433.36): C, 40.19; H, 6.35; N, 9.70. Found: C, 40.32,H, 5.74, N, 9.58.

EXAMPLE 41 DOWEX 50WX4-400 ion-exchange resin salt ofS-[2-[(1-Iminoethyl)amino]ethyl]-2-methyl-L-cysteine

DOWEX® 50WX4-400 (3 g, 1.6 meq/mL, 4.8 meq/g) was washed with deionizedwater (all water used in this experiment was deionized) to pH 6 of thewash. The resin was dried for one hour at room temperature. The productof Example 30 (0.6 g) in 30 mL of water was added to DOWEX resin (0.2g). The suspension was shaken for three hours at ambient temperatureusing an ORBIT™ shaker, then stripped to dryness. This procedure wasrepeated three times with fresh 30 mL of water added after eachconcentration of the reaction mixture. With the final portion of freshwater, the slurry was shaken overnight at ambient temperature

After stripping the reaction to dryness, 15 mL of water were added. Theresin was filtered and washed three times with additional 15 mL ofwater. The filtrate was concentrated (several drops of acetic acid wereadded) and dried under vacuum giving 0.4 g of starting SC-84250 whichwas confirmed by ¹H NMR (D₂O). The loaded resin was dried at ambienttemperature on the bench, followed by 1 hour under vacuum giving 0.3 gof loaded resin. A sample of this resin and a sample of unreacted washedDOWEX 50WX4-400 were submitted for nitrogen combustion analysis: Theresults for the untreated resin were % of N is 0%; for the loaded resin% of N is 9.72%.

EXAMPLE 42 Potassium hydrogen sulfate acid salt ofS-[2-[(1-Iminoethyl)amino]ethyl]-2-methyl-L-cysteine

The title material was prepared by the method of Example 33 from 0.001mole of KHSO₄ and the product of Example 30.

Analysis calculated for C₈H₁₇N₃O₂S+KHSO₄+2H₂O: C, 24.75; H, 5.68; N,10.90; S, 16.00. Found: C, 24.54; H, 5.66; N, 10.73, S, 16.38.

EXAMPLE 43 Potassium hydrogen sulfate acid salt ofS-[2-[(1-Iminoethyl)amino]ethyl]-2-methyl-L-cysteine

The title material was prepared by the method of Example 33 from 0.001mole of KHSO₄ and the product of Example 30.

EXAMPLE 44 Hydrogen sulfate acid salt ofS-[2-[(1-Iminoethyl)amino]ethyl]-2-methyl-L-cysteine

To a stirred 10 mL solution of the product of Example 30 was added 2 mL0.505 N H₂SO₄. After stirring for 2 hours, the solution was concentratedunder vacuum. The resulting salt was dissolved in 10 mL of water andlyophilized.

Analysis calculated for C₈H₁₇N₃O₂S+0.5H₂SO₄+1.5H₂O: C, 32.58; H, 7.23;N, 14.75; S, 16.42. Found: C, 32.53; H, 7.17; N, 14.23, S, 16.28.

EXAMPLE 45 Glycerate salt ofS-[2-[(1-Iminoethyl)amino]ethyl]-2-methyl-L-cysteine

S-Glyceric acid was prepared from its calcium salt by stirring for 4hours with Dowex® 50W resin in its acid form. The resin was filtered andwashed with H₂O. The resulting filtrate was concentrated and dried undervacuum.

Analysis calculated for C₃H₆O₄: C, 31.31, H, 6.13. Found: C, 31.29, H,6.19.

The method described in Example 33 was used to prepare the S-glycericacid salt from the product of Example 30 starting with 0.001 mole ofS-glyceric acid.

Analysis calculated for C₁₁H₂₃N₃O₆S+1.5H₂O: C, 37.49; H, 7.44; N, 11.92;S, 9.10. Found: C, 37.49; H, 7.31; N, 11.73; S, 9.22.

EXAMPLE 46 Malate salt ofS-[2-[(1-Iminoethyl)amino]ethyl]-2-methyl-L-cysteine

The method described in Example 33 was used to prepare the malic acidsalt from the product of Example 30 starting with 0.001 mole of malicacid.

Analysis calculated for C₈H₁₇N₃O₂S+1.33H₂O+C₄H₆O₅: C, 38.20; H, 6.85; N,11.15. Found: C, 38.37; H, 6.51; N, 11.09.

EXAMPLE 47 Hemi-malate salt ofS-[2-[(1-Iminoethyl)amino]ethyl]-2-methyl-L-cysteine

The method described in Example 33 was used to prepare the malic acidsalt from the product of Example 30 starting with 0.0005 mole of malicacid.

Analysis calculated for C₈H₁₇N₃O₂S+1.75H₂O+0.5 C₄H₆O₅: C, 37.92; N,13.22. Found: C, 37.92; H, 7.88; N, 13.03.

EXAMPLE 48 Potassium dihydrogen phosphate salt ofS-[2-[(1-Iminoethyl)amino]ethyl]-2-methyl-L-cysteine

The method described in Example 33 was used to prepare the title saltfrom the product of Example 30.

Analysis calculated for C₈H₁₇N₃O₂S+KH₂PO₄+2.5H₂O+0.66 HOAc: C, 25.44; H,6.10; N, 9.55. Found: C, 25.27; H, 5.95; N, 9.80.

EXAMPLE 49 Sodium dihydrogen phosphate salt ofS-[2-[(1-Iminoethyl)amino]ethyl]-2-methyl-L-cysteine

The method described in Example 33 was used to prepare the title saltfrom the product of Example 30.

Analysis calculated for C₈H₁₇N₃O₂S+NaH₂PO₄+2H₂O+0.3 HOAc: C, 26.26; H,6.20; N, 10.68. Found: C, 26.57; H, 6.25; N, 10.72.

EXAMPLE 50 Calcium dihydrogen phosphate salt ofS-[2-[(1-Iminoethyl)amino]ethyl]-2-methyl-L-cysteine

The method described in Example 33 was used to prepare the title saltfrom the product of Example 30.

Analysis calculated for C₈H₁₇N₃O₂S+2 NaH₂PO₄+2H₂O: C, 19.40; H, 5.09; N,8.48. Found: C, 19.34; H, 5.10; N, 8.56.

EXAMPLE 51 Calcium phosphate salt ofS-[2-[(1-Iminoethyl)amino]ethyl]-2-methyl-L-cysteine

The method described in Example 33 was used to prepare the title saltfrom the product of Example 30.

Analysis calculated for C₈H₁₇N₃O₂S+Ca(H₂PO₄)₂+0.2 HOAc: C, 19.96, H,4.35; N,8.31. Found: C, 20.14; H, 5.73; N, 8.80.

EXAMPLE 52 Calcium hydrogen phosphate salt ofS-[2-[(1-Iminoethyl)amino]ethyl]-2-methyl-L-cysteine

The method described in Example 33 was used to prepare the title saltfrom the product of Example 30.

Analysis calculated for C₈H₁₇N₃O₂S+CaHPO₄+2.2 HCl+H₂O: C, 21.18; H,4.93; N, 9.26. Found: C, 21.20; H, 5.28; N, 9.37.

EXAMPLE 53 Calcium phosphate, tribasic salt ofS-[2-[(1-Iminoethyl)amino]ethyl]-2-methyl-L-cysteine (1.62:1)

The method described in Example 33 was used to prepare the title saltfrom the product of Example 30.

Analysis calculated for C₈H₁₇N₃O₂S+Ca₃ (PO₄)₂+HOAc+3H₂O: C, 14.37; H,2.77; N, 5.03. Found: C, 14.13; H, 3.01; N, 4.71.

EXAMPLE 54 Calcium phosphate, tribasic salt ofS-[2-[(1-Iminoethyl)amino]ethyl]-2-methyl-L-cysteine (1:1)

The method described in Example 33 was used to prepare the title saltfrom the product of Example 30.

Analysis calculated for C₈H₁₇N₃O₂S.Ca₃ (PO₄)₂+2.2 HCl+2H₂O: C, 14.88; H,3.62; N, 6.51. Found: C, 15.09; H, 3.85; N, 6.23.

EXAMPLE 55 Bio-Rex® 70 salt ofS-[2-[(1-Iminoethyl)amino]ethyl]-2-methyl-L-cysteine

The method described in Example 41 was used to prepare the Bio-Rex® 70salt of the neutral form of the product of Example 1. The results forthe untreated resin were % of N is 0%; for the loaded resin % of N is7.93%.

EXAMPLE 56 IPR (amberlite)-69 salt ofS-[2-[(1-Iminoethyl)amino]ethyl]-2-methyl-L-cysteine

The IPR-69 salt of the neutral form of the product of Example 1 wasprepared in the same manner as described in Example 41 except the resinwas treated first with 1 N HCl to convert it to H+form. This resin is apolystyrenedivinylbenzene sulfonic acid resin as is the Dowex-50.However, it is GMP quality but covers a broader mesh size. It is lesscolored than the Dowex. After the washings and prior to loading thecompound, the resin was slurried in H₂O and the fine particles that roseto the top were decanted. From this reaction was recovered 4.9 g ofsalt. 7.69% N (or 0.401 g of SC-84250/g of resin).

EXAMPLE 57 IPR (amberlite)-69 salt ofS-[2-[(1-Iminoethyl)amino]ethyl]-2-methyl-L-cysteine

The IPR-64 salt the neutral form of the product of Example 1 wasprepared in the same manner as described in Example 41 with thedecanting of fines as described in Example 56. This resin is the same asthe Bio-Rex 70 with the exception that is GMP quality. The onlyvariation was after shaking overnight the resin was shaken an additionaltwo times with strips in between. From this reaction was recovered 4.3g. 6.20% N (0.346 g of compound/g of resin

EXAMPLE 58 Preparation of monohydrochloride from the dihydrochloridesalt of S-[2-[(1-Iminoethyl)amino]ethyl]-2-methyl-L-cysteine

Dissolve ˜120 mg of the product of Example 1 in 3 ml of DMF. Add 1 ml ofpropylene oxide and stir. The product will precipitate. Wash with ether.Dissolve product in water and freeze dry. Table 1 shows the elementalanalysis.

EXAMPLE 59 Preparation of monosubstituted salts ofS-12-[(1-Iminoethyl)amino]ethyl]-2-methyl-L-cysteine salts by AgClprecipitation

Dissolve the product of Example 58 in water. Add a stoichiometric amountof a silver salt. Filter the solids. Freeze dry the remaining solution.Table 2 shows the elemental analyses where n represents the moles ofwater.

EXAMPLE 60 Preparation of phosphate salts ofS-[2-[(1-Iminoethyl)amino]ethyl]-2-methyl-L-cysteine salts by AgClprecipitation

Dissolve the product of Example 1 in water. Add two moles of Ag₃PO₄ permole of product of Example 1 and mix. Filter out the solids and freezedry the resulting solution. Analyze the resulting material, and adjustthe phosphate content with H₃PO₄. Table 3 shows the elemental analyseswhere x represents the moles of phosphoric acid.

EXAMPLE 61 Preparation of mixed salts fromS-[2-[(1-Iminoethyl)amino]ethyl]-2-methyl-L-cysteine monohydrochloride

Dissolve the product of Example 58 in water. Add a stoichiometric amountof reagent (R). Table 4 shows the elemental analyses where x representsthe moles of R.

EXAMPLE 62 Preparation of mixed salts fromS-[2-[(1-Iminoethyl)amino]ethyl]-2-methyl-L-cysteine dihydrochloride

Dissolve the product of Example 1 in water. Add base until the pH is 6.Table 5 shows the elemental analyses.

EXAMPLE 63 Preparation of zinc salts fromS-[2-[(1-Iminoethyl)amino]ethyl]-2-methyl-L-cysteine dihydrochloride

Dissolve the product of Example 1 in water. Mix with excess zinc oxide.Filter and freeze dry the resulting solution. Table 6 shows theelemental analysis.

EXAMPLE 64 Preparation of mixed salts fromS-[2-[(1-Iminoethyl)amino]ethyl]-2-methyl-L-cysteine neutral compound

Dissolve the neutral form of the product of Example 20 in water. Mixwith desired reagent and freeze dry. Table 7 shows the elementalanalyses where x represents the moles of MA, metal cation and counteranion.

EXAMPLE 65 Preparation of salts fromS-[2-[(1-Iminoethyl)amino]ethyl]-2-methyl-L-cysteine neutral compound

The following Table 8 lists salts and their elemental analyses preparedfrom the product of Example 1 by one of the variety of methods describedin this application. Table 8 illustrates the elemental analysis of thesesalts where D representsS-[2-[(1-Iminoethyl)amino]ethyl]-2-methyl-L-cysteine with the formula ofC₈H₁₇N₃O₂S and A the empirical formula of the indicated acid and orcounter ion source.

TABLE 1 Calculated Found Formula C H N Cl C H N Cl C₈H₁₇N₃O₂S(HCl)(H₂O)35.10 7.36 15.35 12.95 35.06 7.53 14.90 13.07

TABLE 2 Calculated Found Acid(R) Formula C H N S C H N S

Methane Sulfonic C₈H₁₇N₃O₂S (CH₄SO₃) (H₂O) 32.42 6.95 12.60 19.23 32.097.06 12.36 19.67

Toluene Sulfonic C₈H₁₇N₃O₂S C₇H₈O₃S (H₂O) 43.99 6.65 10.26 15.66 43.836.75 9.91 14.5

Lactic C₈H₁₇N₃O₂S C₃H₆O_(3 (H) ₂O) 40.36 7.70 12.83 9.79 40.79 7.84 12.69.68 HNO₃ C₈H₁₇N₃O₂S 31.99 6.71 18.66 10.67 32.32 5.79 18.32 — NitricHNO₃ (H₂O)

Acetic C₈H₁₇N₃O₂S C₂H₄O₂ 1.5(H₂O) 39.20 7.90 13.72 10.46 39.19 7.1513.53 —

Benzoic C₈H₁₇N₃O₂S C₇H₆O₂ 1.5(H₂O) 48.90 7.11 11.40 8.70 48.93 7.4511.74 —

Pamoic C₈H₁₇N₃O₂S .5(C₂₃H₁₆O₆) 1.5(H₂O) 53.17 6.41 9.54 7.28 53.65 6.389.92 6.92

TABLE 3 Calculated Found Formula C H N P C H N PC₈H₁₇N₃O₂S(H₃PO₄)1.5(H₂O) 27.91 6.73 12.20 9.00 27.74 6.03 12.12 8.73C₈H₁₇N₃O₂S2(H₃PO₄)2(H₂O) 21.29 6.03 9.31 13.73 20.89 5.64 9.3 13.67

TABLE 4 Formula Calculated Found (R) C H N Cl C H N Cl CaCl₂ C₈H₁₇N₃O₂S28.41 6.26 12.42 20.96 28.42 6.3 12.26 19.6 .5(CaCl₂) 1.5(H₂O) (HCl)

Lactic Acid C₈H₁₇N₃O₂S C₃H₆O₃ HCl 38.2 6.99 12.15 10.25 38.32 7.16 12.2310.81

Succinic Acid C₈H₁₇N₃O₂S .5(C₄H₆O₄) HCl .5(H₂O) 37.09 6.85 12.98 10.9537.22 6.68 13.08 11.45

TABLE 5 Calculated Found Formula C H N M Cl C H N M ClC₈H₁₆N₃O₂SLi2(HCl)2(H₂O) 28.75 6.64 12.57 2.08 21.22 28.05 6.58 12.342.14 21.59 C₈H₁₆N₃O₂SNa2(HCl)1.5(H₂O) 28.16 6.20 12.31 — 20.78 27.716.10 12.47 — 22.20 C₈H₁₆N₃O₂SK2(HCl)2(H₂O) 26.23 6.05 11.47 — 19.3625.75 5.60 11.50 — 21.13

TABLE 6 Formula/ Calculated Found References C H N Cl Zn C H N Cl ZnC₈H₁₇N₃O₂S2(HCl)ZnO 25.72 5.13 11.25 18.98 17.50 25.98 4.52 11.57 20.5417.13

TABLE 7 Measured Found Formula C H N C H N C₈H₁₇N₃O₂S(NaCl)2(H2O) 30.626.75 13.39 30.93 6.39 13.27 C₈H₁₇N₃O₂S0.5(CaCl₂)1.5(H₂O) 31.84 6.6813.92 31.59 6.57 13.73 C₈H₁₇N₃O₂S(NaCH₃SO₃)2(H₂O) 28.95 6.48 11.25 28.916.06 10.99

TABLE 8 Calculated Found Acid/Formula C H N C H N L-Tartaric Acid D *0.6A *0.75 H₂O 38.59 6.91 12.98 38.67 6.94 12.65 D *1.0 A *0.5 H₂O 38.096.39 11.10 38.04 6.43 11.38 D *1.2 A *2.0 H₂O 32.48 5.75 8.88 32.76 5.508.96 D *1.1 A *1.5 H₂O 34.37 5.93 9.70 34.51 5.81 9.69 D-Tartaric Acid D*0.5 A *0.5 H₂O 39.59 6.98 13.85 39.27 7.09 13.34 D *1.0 A *1.0 H₂O37.20 6.50 10.85 37.85 6.45 10.43 (R)-(−)-Mandelic Acid D *1.0 A *2.5H₂O 49.99 7.87 10.93 49.90 6.92 10.83 (S)-(+)-Mandelic Acid D *1.0 A*2.75 H₂O 49.41 7.90 10.86 49.38 8.23 10.78 Citric Acid D *0.4 A *0.8H₂O 40.22 7.08 14.23 40.96 6.87 13.26 D *1.0 A *0.5 H₂O *0.1 40.19 6.359.70 40.32 5.74 9.58 HOAc *.015 EtOH S S Mucic Acid D *0.5 A *2.0 H₂O36.66 7.27 11.66 36.46 7.12 11.24 D *0.6 A *1.0 H₂O 40.73 6.84 12.959.89 38.09 7.13 12.03 9.30 Maleic Acid D *0.5 A *1.5 H₂O 39.46 7.2913.81 39.64 6.98 13.01 Malonic Acid D *0.5 A *1.5 H₂O 38.24 7.34 14.0838.03 7.37 13.89 Benzoic Acid D *1.1 A *2.75 H₂O 50.81 7.90 11.32 50.546.39 11.12 D *1.0 A *1.5 H₂O 48.90 7.11 11.40 48.93 7.45 11.74 D *2.4 A*1.4 H₂O 55.41 6.41 7.82 55.75 6.51 7.52 Cl M Cl M Hydrochloric Acid D*1.15 A *2.75 H₂O *0.33 36.17 8.49 14.22 13.79 36.24 8.10 13.98 13.51n-PrOH D *1.0 A *1.0 H₂O 35.10 7.36 15.35 12.95 35.06 7.53 14.90 13.07 D*1.05 A *0.8 H₂O 35.35 7.36 15.44 13.65 35.33 7.28 15.45 13.68 D *1.0 A*1.5 H₂O *0.5 28.41 6.26 12.42 20.96 28.42 6.30 12.26 19.60 CaCl2 Li LiD *2.0 A *2.0 H₂O *1.0 28.75 6.64 12.57 21.22 2.08 28.05 6.58 12.3421.59 2.14 Li+ D *2.0 A *1.5 H₂O *1.0 28.16 6.20 12.31 20.78 27.71 6.1012.47 22.20 Na+ D *2.0 A *2.0 H₂O *1.0 K+ 26.23 6.05 11.47 19.36 25.755.60 11.50 21.13 Zn Zn D *2.0 A *0.5 ZnO 25.72 5.13 11.25 18.98 17.5025.98 4.52 11.57 20.54 17.13 D *1.20 A *1.25 H₂O *1.0 36.47 8.34 14.1819.74 8.11 36.44 8.66 14.18 19.84 6.92 Me4N+ Cl− Cl S Cl SMethanesulfonic Acid D *1.0 A *1.25 HCl *0.8 28.80 6.40 11.19 11.8017.08 28.29 6.38 11.16 12.01 17.17 H₂O D *2.0 A *0.75 HCl *5.0 23.106.74 8.08 5.11 18.50 23.03 6.58 8.16 5.18 18.58 H₂O D *1.0 A *1.0 H₂O32.42 6.95 12.60 19.23 32.09 7.06 12.36 19.67 D *1.0A− *2.0 H₂O *1.028.95 6.48 11.25 28.91 6.06 10.99 Na+ D *1.0 A *1.0 Me₄N+ *1.75 37.178.52 13.34 15.72 37.45 7.92 12.81 14.95 H₂O Tosic Acid D *1.0 A *1.5 H₂O45.07 6.82 9.35 45.60 6.55 9.21 D *2.2 A *2.4 H₂O 46.36 6.55 6.93 46.766.01 6.65 D *1.0 A *1.5 HCl *1.0 H₂O 38.81 6.19 9.05 11.46 13.18 38.906.37 8.93 11.57 13.80 D *1.0 *1.0 H₂O 43.99 6.65 10.26 15.66 43.83 6.759.91 14.50 D *1.0 A− *1.0 Me₄N+ *1.0 47.28 7.94 11.61 13.29 47.39 7.9010.40 12.91 H₂O D *1.0 A *10A− *1.0 47.83 6.79 8.58 14.73 47.95 7.258.49 14.77 Me4N+ *1.0 H₂O D *1.2 A *1.0 H₂O 42.74 6.18 9.84 42.94 5.839.34 D *2.1 A *5.0 H₂O 38.57 6.22 6.55 38.87 5.75 6.50 Br Br HydrobromicAcid D *1.1 A *2.25 H₂O 0.3 30.85 7.17 12.55 26.25 30.66 6.95 12.2026.77 HOAc D *0.61 A *1.0 H₂O 32.01 6.04 14.00 26.62 10.68 31.51 6.3313.82 25.71 10.16 Ethanesulfonic Acid D *1.2 A 35.45 6.94 11.96 35.586.98 11.53 D *2.1 A *1.0 H₂O 31.27 6.80 8.97 31.85 6.62 8.45 D-(+)-MalicAcid D *0.55 A *0.4 H₂O 40.80 7.08 13.99 40.66 6.95 13.401-Adamantaneacetic Acid D *0.91 A *3.5 H₂O 53.11 9.53 9.88 53.44 8.8510.00 1-Adamantane-carboxylic Acid D *0.95 A *3.25 H₂O 52.57 9.37 9.9752.61 7.73 9.92 Flavianic Acid D *1.0 A *1.0 H₂O 39.20 4.57 12.70 11.6338.80 3.94 12.35 11.84 1R-(−)-Camphorsulfonic Acid D *1.0 A *0.75 H₂O46.04 7.51 8.95 13.66 46.12 7.44 8.72 13.66 1S-(+)-Camphorsulfonic AcidD *1.0 A *2.0 H₂O 46.48 7.48 9.03 13.79 46.41 7.85 8.74 14.132-Mesitylenesulfonic Acid D *1.0 A *1.25 H₂O 46.19 7.18 9.50 14.51 45.968.54 7.33 14.91 1,5-Naphthalenedisulfonic Acid D *1.0 A *2.0 H₂O 39.865.25 7.35 17.92 39.65 4.25 7.33 18.77 D *0.6 A *1.250 H₂O 40.54 5.9010.13 17.01 40.03 4.60 10.14 17.44 1,2-Ethanedisulfonic Acid D *0.5 A*0.5 H₂O 33.42 6.54 12.99 19.83 33.01 6.29 12.75 18.96 D *0.5 A *1.5 HCl*1.0 H₂O 27.92 6.12 10.85 13.75 16.57 27.84 5.96 11.05 13.76 16.03 D*1.0 A *0.8 HCl *1.5 H₂O 25.79 5.80 9.02 6.09 20.66 26.07 5.76 9.58 6.0819.97 Sulfonacetic Acid D *1.1 A *1.1 H₂O 33.42 5.89 11.69 17.84 31.616.04 10.92 17.35 1,3-Propanedisulfonic Acid D *0.67 A *1.0 HCl *1.2528.96 6.28 10.12 8.54 18.08 28.72 6.32 10.10 8.96 18.12 H₂O D *0.3 A*1.6 HCl *1.25 29.34 6.56 11.40 15.39 14.53 29.17 6.71 11.50 15.48 14.51H₂O L-(+)-Lactic Acid D *1.0 A *1.0 H₂O 40.36 7.70 12.83 9.79 40.79 7.8412.60 9.68 D *1.0 A *1.0 H₂O *1.0 HCl 38.20 6.99 12.15 10.25 38.32 7.1612.23 10.81 Nitric Acid D *1.0 A *1.0 H₂O 31.99 6.71 18.66 32.32 5.7918.32 Acetic Acid D *1.0 A *1.0 H₂O 39.20 7.90 13.72 39.19 7.15 13.53 D*1.0 A *1.3 H₂O 39.67 7.86 13.88 39.96 7.87 13.69 D *1.0 A *0.05 HCl*2.2 37.41 8.01 13.20 0.56 37.30 7.92 13.17 0.41 H₂O (S-Enantiomer)Pamoic Acid D *0.5 A *1.5 H₂O 53.17 6.41 9.54 7.28 53.65 6.38 9.92 6.92P P Phosphoric Acid D *1.0 A *1.5 H₂O 27.21 6.73 12.20 9.00 27.74 6.0312.12 8.73 D *2.0 A *2.0 H₂O 21.29 6.03 9.31 13.73 20.89 5.64 9.30 13.67D *1.0 A− *2.5 H₂O *0.66 25.44 6.10 9.55 25.27 5.95 9.80 HOAc *1.0 K+ D*1.0 A− *2.0 H₂O *0.33 26.26 6.20 10.68 26.57 6.25 10.72 HOAc *1.0 Na+ D*2.0 A− *0.2 H OAc 19.96 4.35 8.31 20.14 5.73 8.80 *1.0 Ca++ D *1.0 A−*2.2 HCl *1.0 21.18 4.93 9.26 21.20 5.28 9.37 H₂O *0.66 HOAc *1.0 Ca++ D*2.0 A−−− *3.0 H₂O *1.0 14.37 2.77 5.03 14.13 3.01 4.71 HOAc *2.0 Ca++ D*2.0 A−−− *2.2 HCl *2.0 14.88 3.62 6.51 15.09 3.85 6.23 H₂O *0.66 HOAc*3.0 Ca++ Succinic Acid D *0.5 A *0.5 H₂O 37.09 6.85 12.98 10.95 37.226.68 13.08 11.45 *1.0 HCl D *0.5 A *0.5 H₂O 39.99 7.40 12.33 40.35 7.1111.76 *1.0 HCl D *1.0 A *1.5 H₂O 39.55 7.19 11.53 39.24 6.04 11.41(S-Enantiomer) Sodium Chloride D *1.0 A *2.0 H₂O 30.62 6.75 13.39 30.936.39 13.27 Calcium Chloride D *0.5 A *1.5 H₂O 31.84 6.68 13.92 31.596.57 13.73 D *0.5 A *2.5 H₂O 29.09 5.19 12.72 28.40 6.35 10.70D-a-Glacturonic Acid D *1.0 A *1.75 H₂O 37.84 7.00 9.57 7.61 37.86 7.109.60 7.61 Sulfuric Acid D *1.0 A− *2.0 H₂O *1.0 24.75 5.68 10.90 16.0024.54 5.66 10.73 16.38 K+ D *0.5 A *1.5 H₂O 32.58 7.23 14.75 16.42 32.537.17 14.23 16.28 D *1.0 A−− *2.0 25.97 6.97 27.25 13.86 26.12 6.44 27.3213.31 CN3H6+ *1.5 H₂O D *1.5 A−− *.0 CN3H6+ 30.50 7.39 23.71 13.57 30.946.71 23.87 13.23 *0.75 H₂O (S)-Glyceric Acid D *0.5 A *1.5 H₂O 37.497.44 11.92 9.10 37.49 7.31 11.73 9.22 L-(−)-Malic Acid D *1.0 A *1.33H₂O 38.20 6.85 11.15 38.37 6.51 11.09 D *0.5 A *1.75 H₂O 37.92 7.4813.22 37.92 7.88 13.03 L-Ascorbic Acid D *1.0 A *2.2 H₂O 38.65 6.81 9.6638.91 6.80 9.43 3-(N-morpholino)propane Sulfonate D *1.0 A *2.5 H₂O38.04 7.87 11.83 38.41 8.12 11.64 L-cysteic Acid D *0.5 A *2.3 H₂O 30.736.71 13.03 30.91 7.05 12.98 (4S)-hydroxy-L-proline D *1.0 A *0.3 H₂O44.89 7.53 15.74 44.29 7.60 15.91 Cyclopropane-1,1- dicarboxylic Acid D*1.0 A *1.3 H₂O 41.88 6.92 11.27 42.02 6.68 10.83 D *0.5 A *1.5 H₂O40.50 7.45 13.48 40.70 7.10 12.98 2,2-dimethylmalonic Acid D *1.0 A *2.2H₂O 39.93 7.58 10.75 39.88 7.37 10.37 D *0.5 A *1.8 H₂O 39.68 7.80 13.2240.10 7.81 13.38 Ethanolamine D *0.5 A *1.3 H₂O 31.73 7.67 14.80 18.7331.41 7.60 15.00 19.12 *2.0 HCl Ethylene diamine D *0.5 A *1.2 H₂O 32.127.92 18.73 18.96 31.90 9.19 18.08 19.11 *2.0 HCl D,L-Aspartic Acid D*1.0 A *1.8 H₂O 37.60 7.20 13.70 37.59 7.66 13.73 *0.4 HOAc D-GlutamicAcid D *1.0 A *1.8 H₂O 39.18 7.45 13.44 39.47 7.52 13.29 *0.3 HOAcSquaric Acid D *1.0 A *0.5 H₂O 42.10 5.89 12.27 9.37 42.29 5.72 12.739.42 D *0.5 A *0.75 H₂O 41.44 6.78 14.50 11.06 41.43 6.56 14.16 10.85Fumaric Acid D *1.0 A *2.5 H₂O *2.5 41.20 8.34 8.48 41.99 8.33 8.58 EtOH1-hydroxy-2-naphthoic Acid D *1.2 A *1.0 H₂O *1.0 54.72 6.85 8.25 54.326.08 8.31 EtOH 1-hydroxy-2- naphthlenesulfonoc Acid D *0.65 A *2.4 H₂O43.42 6.58 10.48 51.01 6.53 10.39 2-Carboxyethyl phosphonic Acid D *1.1A *1.25 H₂O *1.0 29.08 5.87 9.00 29.34 5.83 8.70 CaCl2 D *1.5 A *1.0 H₂O33.15 6.49 9.64 33.17 6.56 9.28 D *1.5 A *0.75 H₂O *1.0 29.65 5.77 8.3029.66 5.71 8.84 LiCl Phosphonoacetic Acid D *0.5 A *2.5 H₂O 32.58 6.8312.66 32.16 6.73 13.33 D *2.0 A *1.0 H₂O 27.86 6.12 8.12 27.76 5.56 8.40Phenyl phosphonic Acid D *1.0 A *0.5 H₂O 43.52 6.52 10.87 43.90 6.7810.12 L-pyroglutamic Acid D *1.0 A *1.2 H₂O 42.20 7.19 15.15 42.35 7.1015.01 HPF₆ D *1.0 A *0.5 H₂O 26.31 4.97 11.23 26.63 5.10 10.64

BIOLOGICAL DATA

Some or all of the following assays are used to demonstrate the nitricoxide synthase inhibitory activity of the invention's compounds as wellas demonstrate the useful pharmacological properties.

Citrulline Assay for Nitric Oxide Synthase

Nitric oxide synthase (NOS) activity can be measured by monitoring theconversion of L-[2,3-³H]-arginine to L-[2,3-³H]-citrulline (Bredt andSnyder, Proc. Natl. Acad. Sci. U.S.A., 87, 682-685, 1990 and Moore etal, J. Med. Chem., 39, 669-672, 1996). Human inducible NOS (hiNOS),human endothelial constitutive NOS (hecNOS) and human neuronalconstitutive NOS (hncNOS) are each cloned from RNA extracted from humantissue. The cDNA for human inducible NOS (hiNOS) is isolated from aλcDNA library made from RNA extracted from a colon sample from a patientwith ulcerative colitis. The cDNA for human endothelial constitutive NOS(hecNOS) is isolated from a λcDNA library made from RNA extracted fromhuman umbilical vein endothelial cells (HUVEC) and the cDNA for humanneuronal constitutive NOS (hncNOS) is isolated from a λcDNA library madefrom RNA extracted from human cerebellum obtained from a cadaver. Therecombinant enzymes are expressed in Sf9 insect cells using abaculovirus vector (Rodi et al, in The Biology of Nitric Oxide, Pt. 4:Enzymology Biochemistry and Immunology; Moncada, S., Feelisch, M.,Busse, R., Higgs, E., Eds.; Portland Press Ltd.: London, 1995; pp447-450). Enzyme activity is isolated from soluble cell extracts andpartially purified by DEAE-Sepharose chromatography. To measure NOSactivity, 10 μL of enzyme is added to 40 μL of 50 mM Tris (pH 7.6) inthe presence or absence of test compounds and the reaction initiated bythe addition of 50 μL of a reaction mixture containing 50 mM Tris (pH7.6), 2.0 mg/mL bovine serum albumin, 2.0 mM DTT, 4.0 mM CaCl₂, 20 μMFAD, 100 μM tetrahydrobiopterin, 0.4 mM NADPH and 60 μL-argininecontaining 0.9 μCi of L-[2,3-³H]-arginine. The final concentration ofL-arginine in the assay is 30 μM. For hecNOS or hncNOS, calmodulin isincluded at a final concentration of 40-100 nM. Following incubation at37° C. for 15 minutes, the reaction is terminated by addition of 400 μLof a suspension (1 part resin, 3 parts buffer) of Dowex 50W X-8 cationexchange resin in a stop buffer containing 10 mM EGTA, 100 mM HEPES, pH5.5 and 1 mM L-citrulline. After mixing the resin is allowed to settleand L-[2,3-³H]-Citrulline formation is determined by counting aliquotsof the supernatant with a liquid scintillation counter. Results arereported in Table 1 as the IC₅₀ values of compounds for hiNOS, hecNOSand hncNOS.

In Vivo Assay

Rats can be treated with an intraperitoneal injection of 1-12.5 mg/kg ofendotoxin (LPS) with or without oral administration of the nitric oxidesynthase inhibitors. Plasma nitrite/nitrate levels can be determined 5hours post-treatment. The results can be used to show that theadministration of the nitric oxide synthase inhibitors decreases therise in plasma nitrite/nitrate levels, a reliable indicator of theproduction of nitric oxide induced by endotoxin.

Raw Cell Nitrite Assay

RAW 264.7 cells can be plated to confluency on a 96-well tissue cultureplate grown overnight (17h) in the presence of LPS to induce NOS. A rowof 3-6 wells can be left untreated and served as controls forsubtraction of nonspecific background. The media can be removed fromeach well and the cells washed twice with Kreb-Ringers-Hepes (25 mM, pH7.4) with 2 mg/ml glucose. The cells are then placed on ice andincubated with 50 μL of buffer containing L-arginine (30μM)+/−inhibitors for 1 h. The assay can be initiated by warming theplate to 37° C in a water bath for 1 h. Production of nitrite byintracellular iNOS will be linear with time. To terminate the cellularassay, the plate of cells can be placed on ice and thenitrite-containing buffer removed and analyzed for nitrite using apreviously published fluorescent determination for nitrite. T. P. Miskoet al, Analytical Biochemistry, 214, 11-16 (1993).

Human Cartilage Explant Assay

Bone pieces are rinsed twice with Dulbecco's Phosphate Buffered Saline(GibcoBRL) and once with Dulbecco's Modified Eagles Medium (GibcoBRL)and placed into a petri dish with phenol red free Minimum EssentialMedium (MEM) (GibcoBRL). Cartilage was cut into small explants ofapproximately 15-45 mg in weight and one or two explants per well areplaced into either 96 or 48 well culture plates with 200-500 μL ofculture media per well. The culture media was either a custommodification of Minimum Essential Medium(Eagle) with Earle's salts(GibcoBRL) prepared without L-Arginine, without L-Glutamine and withoutphenol red or a custom modification of serumless Neuman and Tytell(GibcoBRL) medium prepared without L-arginine, without insulin, withoutascorbic acid, without L-glutamine and without phenol red. Both aresupplemented before use with 100 μM L-Arginine (Sigma), 2 mML-glutamine, 1×HL-1 supplement (BioWhittaker), 50 mg/ml ascorbic acid(Sigma) and 150 pg/ml recombinant human IL-1β (RD Systems) to inducenitric oxide synthase. Compounds are then added in 10 μL aliquots andthe explants incubated at 37° C. with 5% CO₂ for 18-24 hours. The dayold supernatant is then discarded and replaced with fresh culture mediacontaining recombinant human IL-1β and compound and incubated foranother 20-24 hours. This supernatant is analyzed for nitrite with afluorometric assay (Misko et al, Anal. Biochem., 214, 11-16, 1993). Allsamples are done in quadruplicate. Unstimulated controls are cultured inmedia in the absence of recombinant human IL-1β. IC₅₀ values (Table 1)are determined from plotting the percent inhibition of nitriteproduction at six different concentrations of inhibitor.

Table 9 shows examples of biological activity for some of the compoundsof the present invention.

TABLE 9 Biological Activity. Values represent averages across allexperiments and all lots studied. Example hiNOS Human Number of IC₅₀hecNOS IC₅₀ hncNOS IC₅₀ Cartilage IC₅₀ Compound (μM) (μM) (μM) (μM)Example 1 3.1 77 15 0.7 Example 2 4.4 302 58 8.2 Example 3 74 266 86Example 4 197 1100 539 Example 7 3.4 78 17 Example 11 0.9 26 6.0 Example16 7.2 >100 36 0.7 Example 18 12 >100 181 Example 19 12 1080 220

From the foregoing description, one skilled in the art can easilyascertain the essential characteristics of this invention, and withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the invention to adapt it to various usages andconditions.

1. A method for the preparation of a compound or a pharmaceuticallyacceptable salt thereof, the compound having a structure correspondingto Formula 21:

or a salt thereof, wherein: X is selected from the group consisting of—S—, —S(O)—, and —S(O)₂—; R² is selected from the group consisting ofC₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₅ alkoxy-C₁ alkyl, andC₁-C₅ alkylthio-C₁ alkyl; R³⁰ is selected from the group consisting of—H, —OH, —C(O)—R¹⁷, —C(O)—O—R¹⁸, and —C(O)—S—R¹⁹; R¹, R⁵, R⁶, and R⁷independently are selected from the group consisting of —H, halogen,C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, and C₁-C₅ alkoxy-C₁ alkyl; R⁹and R¹⁰ independently are selected from the group consisting of —H,C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, and C₁-C₅ alkoxy-C₁ alkyl;with respect to R¹¹ and R¹²: R¹¹ is selected from the group consistingof —H, —OH, —C(O)—O—R²⁴, and —C(O)—S—R²⁵; and R¹² is selected from thegroup consisting of —H, —OH, —C(O)—O—R²⁶, and —C(O)—S—R²⁷; or R¹¹ is—O—, and R¹² is —C(O)—, wherein R¹¹ and R¹² together with the atoms towhich they are attached form a ring; or R¹¹ is —C(O)—, and R¹² is —O—,wherein R¹¹ and R¹² together with the atoms to which they are attachedform a ring; and R¹³ is C₁ alkyl; R¹⁷, R¹⁸, R¹⁹, R²⁴, R²⁵, R²⁶, R²⁷ andR^(27a) independently are selected from the group consisting of —H andalkyl, which is optionally substituted by one or more moieties selectedfrom the group consisting of cycloalkyl, heterocyclyl, aryl, andheteroaryl; and when any of R¹, R², R⁴, R⁵, R⁶, R⁷, R⁹, R¹⁰, R¹¹, R¹²,R¹³, R¹⁴, R¹⁵, R¹⁶, R¹⁷, R¹⁸, R¹⁹, R²⁴, R²⁵, R²⁵ R²⁶, R²⁷ and R^(27a)independently is a moiety selected from the group consisting of alkyl,alkenyl, alkynyl, alkoxy, alkylthio, cycloalkyl, heterocyclyl, aryl, andheteroaryl, then the moiety is optionally substituted by one or moresubstituent selected from the group consisting of —OH, alkoxy, andhalogen; wherein the method comprises treating a diamine compound havinga structure corresponding to Formula 22:

or a pharmaceutically acceptable salt thereof, with an alkyl acetimidatehaving a structure corresponding to Formula 23:

or a salt thereof, wherein R³¹ is C₁-C₆ alkyl, to produce the compoundcorresponding to Formula
 21. 2. The method of claim 1 wherein R¹¹ isselected from the group consisting of —H and —OH.
 3. The method of claim2 wherein R¹¹ is —H.
 4. The method of claim 2 wherein R¹¹ is —OH.
 5. Themethod of claim 2 wherein R¹³ is methyl or halomethyl.
 6. The method ofclaim 5 wherein R¹³ is methyl.
 7. The method of claim 2 wherein R³¹ isC₁-C₃ alkyl.
 8. The method of claim 7 wherein R³¹ is ethyl.
 9. Themethod of claim 1 wherein the treating of the diamine compound with thealkyl acetimidate compound is performed in the presence of a base. 10.The method of claim 9 wherein the base is selected from the groupconsisting of a hydrazine, a metal sulfide, a metal hydroxide, a metalalkoxide, an amine, a hydroxylamine, a metal amide complex, and a basicresin.
 11. The method of claim 10 wherein the base is a basic resin. 12.The method of claim 11 wherein the basic resin is a polymer-bounddiazabicyclo[4.4.0]dec-2-ene.
 13. The method of claim 10 wherein thebase is an amine.
 14. The method of claim 13 wherein the base isselected from the group consisting of 1,5-diazabicyclo[4.3.0]non-5-ene;1,4-diazabicyclo[2.2.2]octane; and 1,8-diazabicyclo[5.4.0]undec-7-ene.